Optimized anti-cd3 variable regions

ABSTRACT

The present invention is directed to optimized anti-CD3 variable sequences for use in a variety of bispecific formats, including those that utilize scFv components. The invention further relates to nucleic acids encoding for the polypeptide, to vectors comprising the same and to host cells comprising the vector. In another aspect, the invention provides for a pharmaceutical composition comprising the mentioned polypeptide and medical uses of the polypeptide.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 61/778,148, filed Mar. 12, 2013, which is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

Antibody-based therapeutics have been used successfully to treat avariety of diseases, including cancer and autoimmune/inflammatorydisorders. Yet improvements to this class of drugs are still needed,particularly with respect to enhancing their clinical efficacy. Oneavenue being explored is the engineering of additional and novel antigenbinding sites into antibody-based drugs such that a singleimmunoglobulin molecule co-engages two different antigens. Suchnon-native or alternate antibody formats that engage two differentantigens are often referred to as bispecifics. Because the considerablediversity of the antibody variable region (Fv) makes it possible toproduce an Fv that recognizes virtually any molecule, the typicalapproach to bispecific generation is the introduction of new variableregions into the antibody.

A number of alternate antibody formats have been explored for bispecifictargeting (Chames & Baty, 2009, mAbs 1[6]:1-9; Holliger & Hudson, 2005,Nature Biotechnology 23[9]:1126-1136; Kontermann, mAbs 4(2):182 (2012),all of which are expressly incorporated herein by reference). Initially,bispecific antibodies were made by fusing two cell lines that eachproduced a single monoclonal antibody (Milstein et al., 1983, Nature305:537-540). Although the resulting hybrid hybridoma or quadroma didproduce bispecific antibodies, they were only a minor population, andextensive purification was required to isolate the desired antibody. Anengineering solution to this was the use of antibody fragments to makebispecifics. Because such fragments lack the complex quaternarystructure of a full length antibody, variable light and heavy chains canbe linked in single genetic constructs. Antibody fragments of manydifferent forms have been generated, including diabodies, single chaindiabodies, tandem scFv's, and Fab₂ bispecifics (Chames & Baty, 2009,mAbs 1[6]:1-9; Holliger & Hudson, 2005, Nature Biotechnology23[9]:1126-1136; expressly incorporated herein by reference). Whilethese formats can be expressed at high levels in bacteria and may havefavorable penetration benefits due to their small size, they clearrapidly in vivo and can present manufacturing obstacles related to theirproduction and stability. A principal cause of these drawbacks is thatantibody fragments typically lack the constant region of the antibodywith its associated functional properties, including larger size, highstability, and binding to various Fc receptors and ligands that maintainlong half-life in serum (i.e. the neonatal Fc receptor FcRn) or serve asbinding sites for purification (i.e. protein A and protein G).

More recent work has attempted to address the shortcomings offragment-based bispecifics by engineering dual binding into full lengthantibody-like formats (Wu et al., 2007, Nature Biotechnology25[11]:1290-1297; U.S. Ser. No. 12/477,711; Michaelson et al., 2009,mAbs 1[2]:128-141; PCT/US2008/074693; Zuo et al., 2000, ProteinEngineering 13[5]:361-367; U.S. Ser. No. 09/865,198; Shen et al., 2006,J Biol Chem 281[16]:10706-10714; Lu et al., 2005, J Biol Chem280[20]:19665-19672; PCT/US2005/025472; expressly incorporated herein byreference). These formats overcome some of the obstacles of the antibodyfragment bispecifics, principally because they contain an Fc region. Onesignificant drawback of these formats is that, because they build newantigen binding sites on top of the homodimeric constant chains, bindingto the new antigen is always bivalent.

For many antigens that are attractive as co-targets in a therapeuticbispecific format, the desired binding is monovalent rather thanbivalent. For many immune receptors, cellular activation is accomplishedby cross-linking of a monovalent binding interaction. The mechanism ofcross-linking is typically mediated by antibody/antigen immunecomplexes, or via effector cell to target cell engagement. For example,the low affinity Fc gamma receptors (FcγRs) such as FcγRIIa, FcγRIIb,and FcγRIIIa bind monovalently to the antibody Fc region. Monovalentbinding does not activate cells expressing these FcγRs; however, uponimmune complexation or cell-to-cell contact, receptors are cross-linkedand clustered on the cell surface, leading to activation. For receptorsresponsible for mediating cellular killing, for example FcγRIIIa onnatural killer (NK) cells, receptor cross-linking and cellularactivation occurs when the effector cell engages the target cell in ahighly avid format (Bowles & Weiner, 2005, J Immunol Methods 304:88-99,expressly incorporated by reference). Similarly, on B cells theinhibitory receptor FcγRIIb downregulates B cell activation only when itengages into an immune complex with the cell surface B-cell receptor(BCR), a mechanism that is mediated by immune complexation of solubleIgG's with the same antigen that is recognized by the BCR (Heyman 2003,Immunol Lett 88[2]:157-161; Smith and Clatworthy, 2010, Nature ReviewsImmunology 10:328-343; expressly incorporated by reference). As anotherexample, CD3 activation of T-cells occurs only when its associatedT-cell receptor (TCR) engages antigen-loaded MHC on antigen presentingcells in a highly avid cell-to-cell synapse (Kuhns et al., 2006,Immunity 24:133-139). Indeed nonspecific bivalent cross-linking of CD3using an anti-CD3 antibody elicits a cytokine storm and toxicity(Perruche et al., 2009, J Immunol 183[2]:953-61; Chatenoud & Bluestone,2007, Nature Reviews Immunology 7:622-632; expressly incorporated byreference). Thus for practical clinical use, the preferred mode of CD3co-engagement for redirected killing of targets cells is monovalentbinding that results in activation only upon engagement with theco-engaged target. Furthermore, because of the potential for undesiredCD3-mediated toxicities, there is a need in the art for anti-CD3antibodies and derivatives that cross-react with primate CD3 antigen foruse in preclinical toxicology studies.

There are a number of multispecific antibody or antibody-fragmentformats that are in development, relying on one binding site binding toCD3. These include bispecific binding moieties discussed in US20110262439, for example. The present invention provides a number ofoptimized anti-CD3 variable sequences that can be used in bispecificformats, including formats relying on scFv formats, scFv-Fc fusions, andthe like.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to optimized anti-CD3 variablesequences for use in a variety of bispecific formats, including thosethat utilize scFv components. The invention further relates to nucleicacids encoding for the polypeptide, to vectors comprising the same andto host cells comprising the vector. In another aspect, the inventionprovides for a pharmaceutical composition comprising the mentionedpolypeptide and medical uses of the polypeptide.

Accordingly, the present invention provides compositions comprising ananti-CD3 variable region having a sequence comprising a vhCDR1 havingSEQ ID NO:411, a vhCDR2 having SEQ ID NO:413, a vhCDR3 having SEQ IDNO:416, a v1CDR1 having SEQ ID NO:420, a v1CDR2 having SEQ ID NO:425 anda v1CDR3 having SEQ ID NO:430. In some cases, the variable heavy domainof the composition does not have the sequence depicted in SEQ ID NO:1and the variable light domain of the composition does not have thesequence depicted in SEQ ID NO:2.

In further aspects, the anti-CD3 variable region is an scFv with asequence selected from the group consisting of SEQ ID NOs: 4, 8, 16, 20,32, 40, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104, 108,112, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164,168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220,224, 228, 232, 236, 240, 244, 248, 252, 256, 260, 264, 268, 272, 276,280, 284, 288, 292, 296, 300, 304, 308, 312, 316, 320, 324, 328, 332,336, 340, 344, 348, 352, 356, 360, 364, 368, 372, 376, 380, 384, 388,392, 396, 400, 404, 408.

In an additional aspect, the compositions of the invention furthercomprise an Fc region, including dual scFv-Fc, with a first anti-CD30scFV and a second scFv. In some aspects, the second scFv binds to atarget antigen selected from the group consisting of CD5, CD20, CD30,CD33, CD38, CD40, EGFR, EpCAM, Her2, HM1.24.

In a further aspect, the anti-CD3 variable region comprises a variableheavy region comprising a sequence selected from the group consisting ofSEQ ID NOs: 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61,65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125,129, 133, 137, 141, 145, 149, 153, 157, 161, 165, 169, 173, 177, 181,185, 189, 193, 197, 201, 205, 209, 213, 217, 221, 225, 229, 233, 237,241, 245, 249, 253, 257, 261, 265, 269, 273, 277, 281, 285, 289, 293,297, 301, 305, 309, 313, 317, 321, 325, 329, 333, 337, 341, 345, 349,353, 357, 361, 365, 369, 373, 377, 381, 385, 389, 393, 397, 401, 405,409.

In a further aspect, the anti-CD3 variable region comprises a variablelight region comprising a sequence selected from the group consisting ofSEQ ID NOs: 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62,66, 70, 74, 78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126,130, 134, 138, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 182,186, 190, 194, 198, 202, 206, 210, 214, 218, 222, 226, 230, 234, 238,242, 246, 250, 254, 258, 262, 266, 270, 274, 278, 282, 286, 290, 294,298, 302, 306, 310, 314, 318, 322, 326, 330, 334, 338, 342, 346, 350,354, 358, 362, 366, 370, 374, 378, 382, 386, 390, 394, 398, 402, 406,410.

In an additional aspect, the composition is a bispecific IgG, theanti-CD30 variable region is a first scFv and said composition comprisesa second scFv.

In a further aspect, the composition is a MAb-Fv, wherein the variableheavy domain of the anti-CD30 variable region is attached to a firstheavy chain of the mAb-Fv and the variable light chain of the anti-CD30variable region is attached to a second heavy chain of the mAb-Fv.

In an additional aspect, the composition is a multi-scFv.

In an additional aspect, the composition is an-scFv-CH3.

In an additional aspect, the composition is a mAb-scFv.

In an additional aspect, the composition is a mAb-scFv2.

In an additional aspect, the composition is a full-length antibody.

In a further aspect, the composition comprises an Fc domain andcomprises an amino acid substitution. In some aspects, the Fc domain hasaltered binding to a FcγR receptor, including FcγRIIIa and/or FcγRIIb.In this embodiment, if the altered binding is to FcγRIIIa, the saidamino acid substitution is selected from the group consisting of 239D,239E, 236R, 330L, 332D, 332E, 239D/332E, 239D/332E/330L, 267D, 267E,328F, 328R, 267E/328F, 243L 298A and 236R/328R.

In some aspects, the composition comprises an Fc domain with alteredbinding to a FcRn receptor, and comprises an amino acid substitution isselected from the group consisting of 434A, 434S, 428L, 308F, 259I,428L/434S, 259I/308F, 436I/428L, 436I or V/434S, 436V/428L, 252Y,252Y/254T/256E and 259I/308F/428L.

In a further aspect, the composition has a structure selected from thegroup consisting of FIGS. 7A-7M, FIGS. 8B and 8D, FIG. 9B, FIGS. 10A-10Eand FIGS. 11A-E, wherein the Fc domain of said structure comprises a setof corresponding variants selected from FIGS. 12A and 12B. In otheraspects, the composition has a structure selected from the groupconsisting of FIGS. 7A-7M, FIGS. 8B and 8D, FIG. 9B, FIGS. 10A-10E andFIGS. 11A-E, wherein the Fc domain of the structure comprises a set ofcorresponding variants selected from FIG. 13. In other aspects, thecomposition has a structure selected from the group consisting of FIGS.7A-7M, FIGS. 8B and 8D, FIG. 9B, FIGS. 10A-10E and FIGS. 11A-E, whereinthe Fc domain of the structure comprises a set of corresponding variantsselected from FIG. 14.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C. Stability-optimized, humanized anti-CD3 variant scFvs.Substitutions are given relative to the H1_L1.4 scFv sequence Amino acidnumbering is Kabat numbering.

FIGS. 2A-2YY Amino acid sequences of stability-optimized, humanizedanti-CD3 variant scFvs, variable heavy and variable light sequences.(Note also that the first sequence is the histidine tagged version forease of purification). CDRs are underlined. It should be understood thatthe increased stability of the optimized variable and optimized lightchains (as well as the scFv chains) can be attributed to frameworkregions as well as the CDRs. Thus, it should be understood that thedisclosure of the entire variable region includes the disclosure of theframework regions, although they are not separately numbered.

FIGS. 3A-3B. Yields after Ni-NTA purification and melting temperatures(T_(m)) as determined by DSF (Differential Scanning Fluorimetry) ofstability-optimized, humanized anti-CD3 variant scFvs.

FIGS. 4A-4B. Fold improvement (relative to H1_L1.4 scFv) in cell surfacebinding affinities (IC₅₀) of stability-optimized, humanized anti-CD3variant scFvs as determined in a competitive binding experiment usingpurified human T cells from PBMCs.

FIG. 5. Amino acid sequences that can be used as linkers in scFvs andscFv-containing constructs.

FIG. 6 is the sequence of human CD3.

FIG. 7A-7U depicts a wide variety of the multispecific (e.g.heterodimerization) formats in which the optimized anti-CD3 variableregions find use. As a preliminary matter, the structures of FIGS. 7A-7Mall show a fusion partner of a variable region (including scFvs).However, as described herein for fusion proteins, other binding ligandscan take the place of these variable regions, while one remains CD3specific. FIG. 7A shows a dual scFv-Fc format, that, as for allheterodimerization formats herein (e.g. everything except the IgGantibody of FIG. 8C) can include heterodimerization variants such as pIvariants, knobs in holes (KIH, also referred to herein as stericvariants), charge pairs (a subset of steric variants), and SEED body(“strand-exchange engineered domain”; see Klein et al., mAbs 4:6 653-663(2012) and Davis et al, Protein Eng Des Sel 2010 23:195-202) which relyon the fact that the CH3 domains of human IgG and IgA do not bind toeach other. As for all the heterodimeric structures herein, theseheterodimerization variants can be combined, optionally andindependently and in any combination. What is important is that the“strandedness” of the monomer pairs remains intact although variantslisted as “monomer 1” variants in the steric list can be crossed with“monomer 2” variants in the pI list. That is, any set can be combinedwith any other, regardless of which “monomer” list to which they areassociated. FIG. 7B depicts a bispecific IgG, again with the option of avariety of heterodimerization variants. FIG. 7C depicts the “one armed”version of DVD-Ig which utilizes two different variable heavy andvariable light domains. FIG. 7D is similar, except that rather than an“empty arm”, the variable heavy and light chains are on opposite heavychains. FIG. 7E is generally refered as “mAb-Fv”. FIG. 7F depicts amulti-scFv format; as will be appreciated by those in the art, similarto the “A, B, C, D” formats discussed herein, there may be any number ofassociated scFvs (or, for that matter, any other binding ligands orfunctionalities). Thus, FIG. 7F could have 1, 2, 3 or 4 scFvs (e.g. forbispecifics, the scFv could be “cis” or “trans”, or both on one “end” ofthe molecule). FIG. 7G depicts a heterodimeric FabFc with the Fab beingformed by two different heavy chains one containing heavy chain Fabsequences and the other containing light chain Fab sequences. FIG. 7Hdpeicts the “one armed Fab-Fc”, where one heavy chain comprises the Fab.FIG. 7I depicts a “one armed scFv-Fc”, wherein one heavy chain Fccomprises an scFv and the other heavy chain is “empty”. FIG. 7J shows ascFv-CH3, wherein only heavy chain CH3 regions are used, each with theirown scFv. FIG. 7K depicts a mAb-scFv, wherein one end of the moleculeengages an antigen bivalently with a monovalent engagement using an scFvon one of the heavy chains. FIG. 7L depicts the same structure exceptthat both heavy chains comprise an additional scFv, which can eitherbind the same antigen or different antigens. FIG. 7M shows the“CrossMab” structure, where the problem of multiplex formation due totwo different light chains is addressed by switching sequences in theFab portion. FIG. 7N depicts an scFv, FIG. 7O is a “BiTE” or scFv-scFvlinked by a linker as outlined herein, FIG. 7P depicts a DART, FIG. 7Qdepicts a TandAb, and FIG. 7R shows a diabody. FIGS. 7S, 7T and 7Udepict additional alternative scaffold formats that find use in thepresent invention.

FIGS. 8A-8E illustrate mAb-Fv and mAb-Fab immunoglobulin formats. Anative IgG1 antibody is included for reference (FIG. 8A). AlthoughmAb-Fv (FIG. 8B) and mAb-Fab (FIG. 8C) immunoglobulins can beconstructed with homodimeric CH3 domains, preferably they utilizeheterodimeric CH3 domains, as depicted. Bivalent binding to antigen-1(Ag-1) is mediated by the N-terminal VH/VL pairs (Fv-1), whilemonovalent binding to antigen-2 (Ag-2) is mediated by the C-terminalVH/VL pair (Fv-2). Also shown are the Fab-Fv (FIG. 8D) and Fab-Fab (FIG.8E) analogs that bind both antigen-1 and antigen-2 monovalently. As forthe other figures in FIG. 7, FIGS. 7P to 7S can also includeheterodimerization variants, can include heterodimerization variantssuch as pI variants, knobs in holes (KIH, also referred to herein assteric variants), charge pairs, and SEED body structures, optionally inany combination.

FIGS. 9A-9C illustrate the “triple F” format for bispecificimmunoglobulins. FIG. 9A shows a scFv-Fc format, which also finds use inthe present invention with the pI variants of the invention, asdescribed below (and optionally and independently the otherheterodimerization variants). FIG. 9C depicts a more standard bispecificformat, also utilizing the pI variants of the invention (and optionallyand independently the other heterodimerization variants). FIG. 9B showsthe “triple F” format (sometimes also referred to as the “bottle-opener”configuration; (and optionally and independently the otherheterodimerization variants).

FIGS. 10A-10E depict a variety of “higher multispecificity” embodimentsof the “triple F” format. FIG. 10A shows a “triple F” configuration withanother scFv attached to the Fab monomer (this one, along with FIG. 10D,has a greater molecular weight differential as well). FIG. 10B depicts a“triple F” with another scFv attached to the scFv monomer. FIG. 10Cdepicts a “three scFv” format. FIG. 10D depicts an additional Fabattached to the Fab monomer. FIG. 10E depicts a Fab hooked to one of thescFv monomers.

FIGS. 11A-11E show additional varieties of “higher multispecificity”embodiments of the “triple F” format, all with one monomer comprising anscFv. FIG. 11A shows a “Fab-Fv” format with binding to two differentantigens, with FIG. 11B depicting the “Fab-Fv” format with binding to asingle antigen (e.g. bivalent binding to antigen 1). FIGS. 11C and 11Ddepicts a “Fv-Fab” format with similar bivalent or monovalent additionalantigen binding. FIG. 11E depicts one monomer with a CH1-CL attached tothe second scFv.

FIGS. 12A-12B show novel steric variants. As will be understood by thosein the art, the first column of each table represents “corresponding”monomer pairs or “sets”: that is, monomer 1 has 405A and thecorresponding steric variant is 394F.

FIG. 13A-13B depicts heterodimerization variants that find particularuse in the present invention.

FIG. 14 depicts novel pI heterodimerization variants of particular usein the creation of heterodimeric proteins. As will be understood bythose in the art, the first row of each table represents “corresponding”monomer pairs or sets. Note that the I199T and V397M variants are“neutral” (e.g. not for the purposes of altering pI of one of themonomers). Rather, they contribute to the reduction of immunogenicity byhelping preserve a close epitope; e.g. I199T follows pI variants N203Dor Q196K, and V397M follows K392N. Thus these two variants canoptionally and independently be removed from any of the sequences above.

FIG. 15 depicts a matrix of possible combinations of heterodimerizationformats, heterodimerization variants (separated into pI variants andsteric variants (which includes charge pair variants), Fc variants, FcRnvariants and combinations. Legend A are suitable FcRn variants: 434A,434S, 428L, 308F, 259I, 428L/434S, 259I/308F, 436I/428L, 436I or V/434S,436V/428L, 252Y, 252Y/254T/256E and 259I/308F/428L. That is, the dualscFv-Fc format of FIG. 7A can have any of these FcRn variants. Forclarity, as each heavy chain is different, FcRn variants (as well as theFc variants) can reside on one or both monomers. Legend B are suitableFc variants: 236A, 239D, 239E, 332E, 332D, 239D/332E, 267D, 267E, 328F,267E/328F, 236A/332E, 239D/332E/330Y, 239D, 332E/330L, 236R, 328R,236R/328R, 236N/267E, and 299T. (Note, additional suitable Fc variantsare found in FIG. 41 of US 2006/0024298, the figure and legend of whichare hereby incorporated by reference in their entirety). Legend C aresuitable pI variants, and these, for brevity are imported from FIG. 82,again with the understanding that there is a “strandedness” to pIvariants. Legend D are suitable steric variants (including charge pairvariants); again, for brevity are imported from FIG. 80, again with theunderstanding that there is a “strandedness” to steric variants. LegendE reflects the following possible combinations, again, with each variantbeing independently and optionally combined from the appropriate sourceLegend: 1) pI variants plus FcRn variants; 2) pI variants plus Fcvariants; 3) pI variants plus FcRn variants plus Fc variants; 4) stericvariants plus FcRn variants; 5) steric variants plus Fc variants; 6)steric variants plus FcRn variants plus Fc variants; 7) pI variants plussteric variants plus FcRn variants; 8) pI variants plus steric variantsplus Fc variants; 9) pI variants plus steric variants plus FcRn variantsplus Fc variants; and 10) pI variants plus steric variants.

FIG. 16 illustrates the central mAb-Fv immunoglobulin format (sometimesalso referred to as “Central mAb”). A native IgG1 antibody and mAb-Fvbispecific are included for reference. Although central mAb-Fvimmunoglobulins can be constructed with homodimeric CH3 domains,preferably they utilize heterodimeric CH3 domains, as depicted. Bivalentbinding to antigen-1 (Ag-1) is mediated by the N-terminal VH/VL pairs(Fv-1), while monovalent binding to antigen-2 (Ag-2) is mediated by thecentral VH/VL pair (Fv-2). It should be noted that it is also possiblethat trimeric valency can be accomplished, where there are two differentFv-1 molecules in the construct, as described below.

FIGS. 17A-17I depicts a collation of all the vhCDR1-3 and v1CDR1-3sequences useful in the present invention.

FIGS. 18A-18C depict a number of pI variants that can additionally finduse in the present invention, which is a list of all possible reduced pIvariants created from isotypic substitutions of IgG1-4. Shown are the pIvalues for the three expected species as well as the average delta pIbetween the heterodimer and the two homodimer species present when thevariant heavy chain is transfected with IgG1-WT heavy chain.

FIG. 19. List of all possible increased pI variants created fromisotypic substitutions of IgG1-4. Shown are the pI values for the threeexpected species as well as the average delta pI between the heterodimerand the two homodimer species present when the variant heavy chain istransfected with IgG1-WT heavy chain.

FIGS. 20A-20F. Data table of exemplary pI-engineered variants listing:

XenP# the internal reference number Name (HC) heavy chain sequencedesignation SEQ ID NO (HC) corresponding SEQ ID NO of the heavy chainsequence Name (LC) light chain sequence designation SEQ ID NO (LC)corresponding SEQ ID NO of the light chain sequence Calc. pI calculatedpI value for the entire antibody sequence, including heavy and lightchain Fv + constant domains, with the Fv of bevacizumab and the constantdomains as defined in the table #KR number of Lys or Arg residues inIgG1 with the Fv of bevacizumab and the constant domains as defined inthe table Delta KR (vs. change in the number of Lys or Arg residuesrelative WT) to IgG1 wild-type sequence of bevacizumab #DE number of Aspor Glu residues in IgG1 with the Fv of bevacizumab and the constantdomains as defined in the table Delta DE (vs. change in the number ofAsp or Glu acid residues WT) relative to IgG1 wild-type sequence ofbevacizumab Charge state derived from the total number of Lys and Argminus the total number of Asp and Glu residues, assuming a pH of 7 # HCMutations number of mutations in the heavy chain constant vs IgG1 domainas compared to IgG1 # LC Mutations number of mutations in the lightchain constant vs IgG1 domain as compared to IgG1 Total # of totalnumber of mutations in the heavy chain and light Mutations chainconstant domains as compared to IgG1It should be noted that FIGS. 20A-20F have SEQ ID NOs that areassociated with the sequence listing filed in U.S. Ser. No. 13/648,951,and are hereby expressly incorporated by reference.

FIGS. 21A-21B. An additional list of potential heterodimerizationvariants for use in the present invention, optionally and independentlycombined with any variants.

FIG. 22 shows some particular framework regions that confer particularstability, that can be optionally and independently combined with otherframework and CDR regions.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

Bispecific antibodies that incorporate a monovalent binding of human CD3(episilon) are finding use for a variety of treatments. The presentinvention is directed to a variety of new variable sequences that bindto CD3 that display altered properties, including, but not limited to,altered stability and altered binding affinity, in many cases improvedstability and improved binding affinity (although as will be appreciatedby those in the art, in some cases lowered binding affinity can be anadvantage). In addition, these optimized sequences also bind to primateCD3, thus allowing preclinical animal testing.

These variable sequences can be used in scFv formats, such as depictedin FIGS. 7A-7U and FIG. 13, or the variable regions can be used as“traditional” heavy and light variable regions, used in standardmonoclonal antibodies, or in non-scFv multispecific formats. Theinvention further relates to nucleic acids encoding for the polypeptide,to vectors comprising the same and to host cells comprising thevector(s) and to methods of producing the polypeptide. In anotheraspect, the invention provides for a pharmaceutical compositioncomprising the mentioned polypeptide and medical uses of thepolypeptide.

In addition, it should be noted that the pI variants of theheterodimerization variants give an additional benefit for the analyticsand quality control process of bispecific antibodies, as, particularlyin the case of CD3 antibodies, the ability to either eliminate, minimizeand distinguish when homodimers are present is significant. Similarly,the ability to reliably test the reproducibility of the heterodimericprotein production is important.

II. Definitions

In order that the application may be more completely understood, severaldefinitions are set forth below. Such definitions are meant to encompassgrammatical equivalents.

By “ablation” herein is meant a decrease or removal of activity. Thusfor example, “ablating FcγR binding” means the Fc region amino acidvariant has less than 50% starting binding as compared to an Fc regionnot containing the specific variant, with less than 70-80-90-95-98% lossof activity being preferred, and in general, with the activity beingbelow the level of detectable binding in a Biacore assay. Of particularuse in the ablation of FcγR binding is the double variant 236R/328R, and236R and 328R separately as well.

By “ADCC” or “antibody dependent cell-mediated cytotoxicity” as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause lysis of the target cell. ADCC is correlated withbinding to FcγRIIIa; increased binding to FcγRIIIa leads to an increasein ADCC activity.

By “ADCP” or antibody dependent cell-mediated phagocytosis as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause phagocytosis of the target cell.

By “modification” herein is meant an amino acid substitution, insertion,and/or deletion in a polypeptide sequence or an alteration to a moietychemically linked to a protein. For example, a modification may be analtered carbohydrate or PEG structure attached to a protein. By “aminoacid modification” herein is meant an amino acid substitution,insertion, and/or deletion in a polypeptide sequence. For clarity,unless otherwise noted, the amino acid modification is always to anamino acid coded for by DNA, e.g. the 20 amino acids that have codons inDNA and RNA.

By “amino acid substitution” or “substitution” herein is meant thereplacement of an amino acid at a particular position in a parentpolypeptide sequence with a different amino acid. In particular, in someembodiments, the substitution is to an amino acid that is not naturallyoccurring at the particular position, either not naturally occurringwithin the organism or in any organism. For example, the substitutionE272Y refers to a variant polypeptide, in this case an Fc variant, inwhich the glutamic acid at position 272 is replaced with tyrosine. Forclarity, a protein which has been engineered to change the nucleic acidcoding sequence but not change the starting amino acid (for exampleexchanging CGG (encoding arginine) to CGA (still encoding arginine) toincrease host organism expression levels) is not an “amino acidsubstitution”; that is, despite the creation of a new gene encoding thesame protein, if the protein has the same amino acid at the particularposition that it started with, it is not an amino acid substitution.

By “amino acid insertion” or “insertion” as used herein is meant theaddition of an amino acid sequence at a particular position in a parentpolypeptide sequence. For example, −233E or 233E designates an insertionof glutamic acid after position 233 and before position 234.Additionally, −233ADE or A233ADE designates an insertion of AlaAspGluafter position 233 and before position 234.

By “amino acid deletion” or “deletion” as used herein is meant theremoval of an amino acid sequence at a particular position in a parentpolypeptide sequence. For example, E233- or E233# designates a deletionof glutamic acid at position 233. Additionally, EDA233- or EDA233#designates a deletion of the sequence GluAspAla that begins at position233.

By “variant protein” or “protein variant”, or “variant” as used hereinis meant a protein that differs from that of a parent protein by virtueof at least one amino acid modification. Protein variant may refer tothe protein itself, a composition comprising the protein, or the aminosequence that encodes it. Preferably, the protein variant has at leastone amino acid modification compared to the parent protein, e.g. fromabout one to about seventy amino acid modifications, and preferably fromabout one to about five amino acid modifications compared to the parent.As described below, in some embodiments the parent polypeptide, forexample an Fc parent polypeptide, is a human wild type sequence, such asthe Fc region from IgG1, IgG2, IgG3 or IgG4, although human sequenceswith variants can also serve as “parent polypeptides”. The proteinvariant sequence herein will preferably possess at least about 80%identity with a parent protein sequence, and most preferably at leastabout 90% identity, more preferably at least about 95-98-99% identity.Variant protein can refer to the variant protein itself, compositionscomprising the protein variant, or the DNA sequence that encodes it.Accordingly, by “antibody variant” or “variant antibody” as used hereinis meant an antibody that differs from a parent antibody by virtue of atleast one amino acid modification, “IgG variant” or “variant IgG” asused herein is meant an antibody that differs from a parent IgG (again,in many cases, from a human IgG sequence) by virtue of at least oneamino acid modification, and “immunoglobulin variant” or “variantimmunoglobulin” as used herein is meant an immunoglobulin sequence thatdiffers from that of a parent immunoglobulin sequence by virtue of atleast one amino acid modification. “Fc variant” or “variant Fc” as usedherein is meant a protein comprising an amino acid modification in an Fcdomain. The Fc variants of the present invention are defined accordingto the amino acid modifications that compose them. Thus, for example,N434S or 434S is an Fc variant with the substitution serine at position434 relative to the parent Fc polypeptide, wherein the numbering isaccording to the EU index. Likewise, M428L/N434S defines an Fc variantwith the substitutions M428L and N434S relative to the parent Fcpolypeptide. The identity of the WT amino acid may be unspecified, inwhich case the aforementioned variant is referred to as 428L/434S. It isnoted that the order in which substitutions are provided is arbitrary,that is to say that, for example, 428L/434S is the same Fc variant asM428L/N434S, and so on. For all positions discussed in the presentinvention that relate to antibodies, unless otherwise noted, amino acidposition numbering is according to the EU index. The EU index or EUindex as in Kabat or EU numbering scheme refers to the numbering of theEU antibody (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85,hereby entirely incorporated by reference.) The modification can be anaddition, deletion, or substitution. Substitutions can include naturallyoccurring amino acids and, in some cases, synthetic amino acids.Examples include U.S. Pat. No. 6,586,207; WO 98/48032; WO 03/073238;US2004-0214988A1; WO 05/35727A2; WO 05/74524A2; J. W. Chin et al.,(2002), Journal of the American Chemical Society 124:9026-9027; J. W.Chin, & P. G. Schultz, (2002), ChemBioChem 11:1135-1137; J. W. Chin, etal., (2002), PICAS United States of America 99:11020-11024; and, L.Wang, & P. G. Schultz, (2002), Chem. 1-10, all entirely incorporated byreference.

As used herein, “protein” herein is meant at least two covalentlyattached amino acids, which includes proteins, polypeptides,oligopeptides and peptides. The peptidyl group may comprise naturallyoccurring amino acids and peptide bonds, or synthetic peptidomimeticstructures, i.e. “analogs”, such as peptoids (see Simon et al., PNAS USA89(20):9367 (1992), entirely incorporated by reference). The amino acidsmay either be naturally occurring or synthetic (e.g. not an amino acidthat is coded for by DNA); as will be appreciated by those in the art.For example, homo-phenylalanine, citrulline, ornithine and noreleucineare considered synthetic amino acids for the purposes of the invention,and both D- and L-(R or S) configured amino acids may be utilized. Thevariants of the present invention may comprise modifications thatinclude the use of synthetic amino acids incorporated using, forexample, the technologies developed by Schultz and colleagues, includingbut not limited to methods described by Cropp & Shultz, 2004, TrendsGenet. 20(12):625-30, Anderson et al., 2004, Proc Natl Acad Sci USA 101(2):7566-71, Zhang et al., 2003, 303(5656):371-3, and Chin et al., 2003,Science 301(5635):964-7, all entirely incorporated by reference. Inaddition, polypeptides may include synthetic derivatization of one ormore side chains or termini, glycosylation, PEGylation, circularpermutation, cyclization, linkers to other molecules, fusion to proteinsor protein domains, and addition of peptide tags or labels.

By “residue” as used herein is meant a position in a protein and itsassociated amino acid identity. For example, Asparagine 297 (alsoreferred to as Asn297 or N297) is a residue at position 297 in the humanantibody IgG1.

By “Fab” or “Fab region” as used herein is meant the polypeptide thatcomprises the VH, CHL VL, and CL immunoglobulin domains. Fab may referto this region in isolation, or this region in the context of a fulllength antibody, antibody fragment or Fab fusion protein. By “Fv” or “Fvfragment” or “Fv region” as used herein is meant a polypeptide thatcomprises the VL and VH domains of a single antibody.

By “IgG subclass modification” or “isotype modification” as used hereinis meant an amino acid modification that converts one amino acid of oneIgG isotype to the corresponding amino acid in a different, aligned IgGisotype. For example, because IgG1 comprises a tyrosine and IgG2 aphenylalanine at EU position 296, a F296Y substitution in IgG2 isconsidered an IgG subclass modification.

By “non-naturally occurring modification” as used herein is meant anamino acid modification that is not isotypic. For example, because noneof the IgGs comprise a serine at position 434, the substitution 434S inIgG1, IgG2, IgG3, or IgG4 (or hybrids thereof) is considered anon-naturally occurring modification.

By “amino acid” and “amino acid identity” as used herein is meant one ofthe 20 naturally occurring amino acids that are coded for by DNA andRNA.

By “effector function” as used herein is meant a biochemical event thatresults from the interaction of an antibody Fc region with an Fcreceptor or ligand. Effector functions include but are not limited toADCC, ADCP, and CDC.

By “IgG Fc ligand” as used herein is meant a molecule, preferably apolypeptide, from any organism that binds to the Fc region of an IgGantibody to form an Fc/Fc ligand complex. Fc ligands include but are notlimited to FcγRIs, FcγRIIs, FcγRIIIs, FcRn, C1q, C3, mannan bindinglectin, mannose receptor, staphylococcal protein A, streptococcalprotein G, and viral FcγR. Fc ligands also include Fc receptor homologs(FcRH), which are a family of Fc receptors that are homologous to theFcγRs (Davis et al., 2002, Immunological Reviews 190:123-136, entirelyincorporated by reference). Fc ligands may include undiscoveredmolecules that bind Fc. Particular IgG Fc ligands are FcRn and Fc gammareceptors. By “Fc ligand” as used herein is meant a molecule, preferablya polypeptide, from any organism that binds to the Fc region of anantibody to form an Fc/Fc ligand complex.

By “Fc gamma receptor”, “FcγR” or “FcqammaR” as used herein is meant anymember of the family of proteins that bind the IgG antibody Fc regionand is encoded by an FcγR gene. In humans this family includes but isnot limited to FcγRI (CD64), including isoforms FcγRIa, FcγRIb, andFcγRIc; FcγRII (CD32), including isoforms FcγRIIa (including allotypesH131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), andFcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (includingallotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIb-NA1and FcγRIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, entirelyincorporated by reference), as well as any undiscovered human FcγRs orFcγR isoforms or allotypes. An FcγR may be from any organism, includingbut not limited to humans, mice, rats, rabbits, and monkeys. Mouse FcγRsinclude but are not limited to FcγRI (CD64), FcγRII (CD32), FcγRIII(CD16), and FcγRIII-2 (CD16-2), as well as any undiscovered mouse FcγRsor FcγR isoforms or allotypes.

By “FcRn” or “neonatal Fc Receptor” as used herein is meant a proteinthat binds the IgG antibody Fc region and is encoded at least in part byan FcRn gene. The FcRn may be from any organism, including but notlimited to humans, mice, rats, rabbits, and monkeys. As is known in theart, the functional FcRn protein comprises two polypeptides, oftenreferred to as the heavy chain and light chain. The light chain isbeta-2-microglobulin and the heavy chain is encoded by the FcRn gene.Unless otherwise noted herein, FcRn or an FcRn protein refers to thecomplex of FcRn heavy chain with beta-2-microglobulin. A variety of FcRnvariants used to increase binding to the FcRn receptor, and in somecases, to increase serum half-life, are discussed in Legend A of FIG.14.

By “parent polypeptide” as used herein is meant a starting polypeptidethat is subsequently modified to generate a variant. The parentpolypeptide may be a naturally occurring polypeptide, or a variant orengineered version of a naturally occurring polypeptide. Parentpolypeptide may refer to the polypeptide itself, compositions thatcomprise the parent polypeptide, or the amino acid sequence that encodesit. Accordingly, by “parent immunoglobulin” as used herein is meant anunmodified immunoglobulin polypeptide that is modified to generate avariant, and by “parent antibody” as used herein is meant an unmodifiedantibody that is modified to generate a variant antibody. It should benoted that “parent antibody” includes known commercial, recombinantlyproduced antibodies as outlined below.

By “Fc fusion protein” or “immunoadhesin” herein is meant a proteincomprising an Fc region, generally linked (optionally through a linkermoiety, as described herein) to a different protein, such as a bindingmoiety to a target protein, as described herein).

By “position” as used herein is meant a location in the sequence of aprotein. Positions may be numbered sequentially, or according to anestablished format, for example the EU index for antibody numbering.

By “target antigen” as used herein is meant the molecule that is boundspecifically by the variable region of a given antibody. A targetantigen may be a protein, carbohydrate, lipid, or other chemicalcompound. A wide number of suitable target antigens are described below.

By “strandedness” in the context of the monomers of the heterodimericproteins of the invention herein is meant that, similar to the twostrands of DNA that “match”, heterodimerization variants areincorporated into each monomer so as to preserve the ability to “match”to form heterodimers. For example, if some pI variants are engineeredinto monomer A (e.g. making the pI higher) then steric variants that are“charge pairs” that can be utilized as well do not interfere with the pIvariants, e.g. the charge variants that make a pI higher are put on thesame “strand” or “monomer” to preserve both functionalities.

By “target cell” as used herein is meant a cell that expresses a targetantigen.

By “variable region” as used herein is meant the region of animmunoglobulin that comprises one or more Ig domains substantiallyencoded by any of the V.kappa., V.lamda., and/or VH genes that make upthe kappa, lambda, and heavy chain immunoglobulin genetic locirespectively.

By “wild type or WT” herein is meant an amino acid sequence or anucleotide sequence that is found in nature, including allelicvariations. A WT protein has an amino acid sequence or a nucleotidesequence that has not been intentionally modified.

Heterodimeric Proteins

The present invention is directed to the generation of multispecific,particularly bispecific binding proteins, and in particular,multispecific antibodies.

Antibodies

The present invention relates to the generation of heterodimericantibodies, generally therapeutic antibodies, through the use of“heterodimerization amino acid variants”. As is discussed below, theterm “antibody” is used generally. Antibodies that find use in thepresent invention can take on a number of formats as described herein,including traditional antibodies as well as antibody derivatives,fragments and mimetics, described below. In general, the term “antibody”includes any polypeptide that includes at least one constant domain,including, but not limited to, CHL CH2, CH3 and CL.

Traditional antibody structural units typically comprise a tetramer.Each tetramer is typically composed of two identical pairs ofpolypeptide chains, each pair having one “light” (typically having amolecular weight of about 25 kDa) and one “heavy” chain (typicallyhaving a molecular weight of about 50-70 kDa). Human light chains areclassified as kappa and lambda light chains. The present invention isdirected to the IgG class, which has several subclasses, including, butnot limited to IgG1, IgG2, IgG3, and IgG4. Thus, “isotype” as usedherein is meant any of the subclasses of immunoglobulins defined by thechemical and antigenic characteristics of their constant regions. Itshould be understood that therapeutic antibodies can also comprisehybrids of isotypes and/or subclasses. For example, as shown herein, thepresent invention covers heterodimers that can contain one or bothchains that are IgG1/G2 hybrids (see SEQ ID NO:6, for example).

The amino-terminal portion of each chain includes a variable region ofabout 100 to 110 or more amino acids primarily responsible for antigenrecognition, generally referred to in the art and herein as the “Fvdomain” or “Fv region”. In the variable region, three loops are gatheredfor each of the V domains of the heavy chain and light chain to form anantigen-binding site. Each of the loops is referred to as acomplementarity-determining region (hereinafter referred to as a “CDR”),in which the variation in the amino acid sequence is most significant.“Variable” refers to the fact that certain segments of the variableregion differ extensively in sequence among antibodies. Variabilitywithin the variable region is not evenly distributed. Instead, the Vregions consist of relatively invariant stretches called frameworkregions (FRs) of 15-30 amino acids separated by shorter regions ofextreme variability called “hypervariable regions” that are each 9-15amino acids long or longer.

Each VH and VL is composed of three hypervariable regions(“complementary determining regions,” “CDRs”) and four FRs, arrangedfrom amino-terminus to carboxy-terminus in the following order:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

The hypervariable region generally encompasses amino acid residues fromabout amino acid residues 24-34 (LCDR1; “L” denotes light chain), 50-56(LCDR2) and 89-97 (LCDR3) in the light chain variable region and aroundabout 31-35B (HCDR1; “H” denotes heavy chain), 50-65 (HCDR2), and 95-102(HCDR3) in the heavy chain variable region; Kabat et al., SEQUENCES OFPROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991) and/or thoseresidues forming a hypervariable loop (e.g. residues 26-32 (LCDR1),50-52 (LCDR2) and 91-96 (LCDR3) in the light chain variable region and26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chainvariable region; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917.Specific CDRs of the invention are described below.

Throughout the present specification, the Kabat numbering system isgenerally used when referring to a residue in the variable domain(approximately, residues 1-107 of the light chain variable region andresidues 1-113 of the heavy chain variable region) (e.g, Kabat et al.,supra (1991)).

The CDRs contribute to the formation of the antigen-binding, or morespecifically, epitope binding site of antibodies. “Epitope” refers to adeterminant that interacts with a specific antigen binding site in thevariable region of an antibody molecule known as a paratope. Epitopesare groupings of molecules such as amino acids or sugar side chains andusually have specific structural characteristics, as well as specificcharge characteristics. A single antigen may have more than one epitope.

The epitope may comprise amino acid residues directly involved in thebinding (also called immunodominant component of the epitope) and otheramino acid residues, which are not directly involved in the binding,such as amino acid residues which are effectively blocked by thespecifically antigen binding peptide; in other words, the amino acidresidue is within the footprint of the specifically antigen bindingpeptide.

Epitopes may be either conformational or linear. A conformationalepitope is produced by spatially juxtaposed amino acids from differentsegments of the linear polypeptide chain. A linear epitope is oneproduced by adjacent amino acid residues in a polypeptide chain.Conformational and nonconformational epitopes may be distinguished inthat the binding to the former but not the latter is lost in thepresence of denaturing solvents.

An epitope typically includes at least 3, and more usually, at least 5or 8-10 amino acids in a unique spatial conformation. Antibodies thatrecognize the same epitope can be verified in a simple immunoassayshowing the ability of one antibody to block the binding of anotherantibody to a target antigen, for example “binning.”

In some embodiments, the antibodies are full length. By “full lengthantibody” herein is meant the structure that constitutes the naturalbiological form of an antibody, including variable and constant regions,including one or more modifications as outlined herein.

Alternatively, the antibodies can be a variety of structures, including,but not limited to, antibody fragments, monoclonal antibodies,bispecific antibodies, minibodies, domain antibodies, syntheticantibodies (sometimes referred to herein as “antibody mimetics”),chimeric antibodies, humanized antibodies, antibody fusions (sometimesreferred to as “antibody conjugates”), and fragments of each,respectively.

Antibody Fragments

In one embodiment, the antibody is an antibody fragment. Of particularinterest are antibodies that comprise Fc regions, Fc fusions, and theconstant region of the heavy chain (CH1-hinge-CH2-CH3), again alsoincluding constant heavy region fusions.

Specific antibody fragments include, but are not limited to, (i) the Fabfragment consisting of VL, VH, CL and CH1 domains, (ii) the Fd fragmentconsisting of the VH and CH1 domains, (iii) the Fv fragment consistingof the VL and VH domains of a single antibody; (iv) the dAb fragment(Ward et al., 1989, Nature 341:544-546, entirely incorporated byreference) which consists of a single variable, (v) isolated CDRregions, (vi) F(ab′)2 fragments, a bivalent fragment comprising twolinked Fab fragments (vii) single chain Fv molecules (scFv), wherein aVH domain and a VL domain are linked by a peptide linker which allowsthe two domains to associate to form an antigen binding site (Bird etal., 1988, Science 242:423-426, Huston et al., 1988, Proc. Natl. Acad.Sci. U.S.A. 85:5879-5883, entirely incorporated by reference), (viii)bispecific single chain Fv (WO 03/11161, hereby incorporated byreference) and (ix) “diabodies” or “triabodies”, multivalent ormultispecific fragments constructed by gene fusion (Tomlinson et. al.,2000, Methods Enzymol. 326:461-479; WO94/13804; Holliger et al., 1993,Proc. Natl. Acad. Sci. U.S.A. 90:6444-6448, all entirely incorporated byreference). The antibody fragments may be modified. For example, themolecules may be stabilized by the incorporation of disulphide bridgeslinking the VH and VL domains (Reiter et al., 1996, Nature Biotech.14:1239-1245, entirely incorporated by reference).

Chimeric and Humanized Antibodies

In some embodiments, the scaffold components can be a mixture fromdifferent species. As such, if the protein is an antibody, such antibodymay be a chimeric antibody and/or a humanized antibody. In general, both“chimeric antibodies” and “humanized antibodies” refer to antibodiesthat combine regions from more than one species. For example, “chimericantibodies” traditionally comprise variable region(s) from a mouse (orrat, in some cases) and the constant region(s) from a human. “Humanizedantibodies” generally refer to non-human antibodies that have had thevariable-domain framework regions swapped for sequences found in humanantibodies. Generally, in a humanized antibody, the entire antibody,except the CDRs, is encoded by a polynucleotide of human origin or isidentical to such an antibody except within its CDRs. The CDRs, some orall of which are encoded by nucleic acids originating in a non-humanorganism, are grafted into the beta-sheet framework of a human antibodyvariable region to create an antibody, the specificity of which isdetermined by the engrafted CDRs. The creation of such antibodies isdescribed in, e.g., WO 92/11018, Jones, 1986, Nature 321:522-525,Verhoeyen et al., 1988, Science 239:1534-1536, all entirely incorporatedby reference. “Backmutation” of selected acceptor framework residues tothe corresponding donor residues is often required to regain affinitythat is lost in the initial grafted construct (U.S. Pat. No. 5,530,101;U.S. Pat. No. 5,585,089; U.S. Pat. No. 5,693,761; U.S. Pat. No.5,693,762; U.S. Pat. No. 6,180,370; U.S. Pat. No. 5,859,205; U.S. Pat.No. 5,821,337; U.S. Pat. No. 6,054,297; U.S. Pat. No. 6,407,213, allentirely incorporated by reference). The humanized antibody optimallyalso will comprise at least a portion of an immunoglobulin constantregion, typically that of a human immunoglobulin, and thus willtypically comprise a human Fc region. Humanized antibodies can also begenerated using mice with a genetically engineered immune system. Roqueet al., 2004, Biotechnol. Prog. 20:639-654, entirely incorporated byreference. A variety of techniques and methods for humanizing andreshaping non-human antibodies are well known in the art (See Tsurushita& Vasquez, 2004, Humanization of Monoclonal Antibodies, MolecularBiology of B Cells, 533-545, Elsevier Science (USA), and referencescited therein, all entirely incorporated by reference). Humanizationmethods include but are not limited to methods described in Jones etal., 1986, Nature 321:522-525; Riechmann et al., 1988; Nature332:323-329; Verhoeyen et al., 1988, Science, 239:1534-1536; Queen etal., 1989, Proc Natl Acad Sci, USA 86:10029-33; He et al., 1998, J.Immunol. 160: 1029-1035; Carter et al., 1992, Proc Natl Acad Sci USA89:4285-9, Presta et al., 1997, Cancer Res. 57(20):4593-9; Gorman etal., 1991, Proc. Natl. Acad. Sci. USA 88:4181-4185; O'Connor et al.,1998, Protein Eng 11:321-8, all entirely incorporated by reference.Humanization or other methods of reducing the immunogenicity of nonhumanantibody variable regions may include resurfacing methods, as describedfor example in Roguska et al., 1994, Proc. Natl. Acad. Sci. USA91:969-973, entirely incorporated by reference. In one embodiment, theparent antibody has been affinity matured, as is known in the art.Structure-based methods may be employed for humanization and affinitymaturation, for example as described in U.S. Ser. No. 11/004,590.Selection based methods may be employed to humanize and/or affinitymature antibody variable regions, including but not limited to methodsdescribed in Wu et al., 1999, J. Mol. Biol. 294:151-162; Baca et al.,1997, J. Biol. Chem. 272(16):10678-10684; Rosok et al., 1996, J. Biol.Chem. 271(37): 22611-22618; Rader et al., 1998, Proc. Natl. Acad. Sci.USA 95: 8910-8915; Krauss et al., 2003, Protein Engineering16(10):753-759, all entirely incorporated by reference. Otherhumanization methods may involve the grafting of only parts of the CDRs,including but not limited to methods described in U.S. Ser. No.09/810,510; Tan et al., 2002, J. Immunol. 169:1119-1125; De Pascalis etal., 2002, J. Immunol. 169:3076-3084, all entirely incorporated byreference.

In one embodiment, the antibody is a minibody. Minibodies are minimizedantibody-like proteins comprising a scFv joined to a CH3 domain. Hu etal., 1996, Cancer Res. 56:3055-3061, entirely incorporated by reference.In some cases, the scFv can be joined to the Fc region, and may includesome or the entire hinge region.

Fc Fusion Heterodimeric Proteins

In addition to heterodimeric antibody constructs, the invention furtherprovides Fc fusion heterodimeric proteins that include a novel anti-CD3variable domain (such as an scFv) as one of the binding moieties.However, rather than have the Fc domain of an antibody joined to anotherantibody variable region, the Fc domain can be joined to other moieties,particularly binding moieties such as ligands. That is, one Fc domain ofthe heavy chain may be an anti-CD3 domain as described herein, and theother Fc domain of the heavy chain can be a ligand. By “Fc fusion” asused herein is meant a protein wherein one or more polypeptides isoperably linked to an Fc region. Fc fusion is herein meant to besynonymous with the terms “immunoadhesin”, “Ig fusion”, “Ig chimera”,and “receptor globulin” (sometimes with dashes) as used in the prior art(Chamow et al., 1996, Trends Biotechnol 14:52-60; Ashkenazi et al.,1997, Curr Opin Immunol 9:195-200, both entirely incorporated byreference). An Fc fusion combines the Fc region of an immunoglobulinwith a fusion partner, which in general can be any protein or smallmolecule. Virtually any protein or small molecule may be linked to Fc togenerate an Fc fusion. Protein fusion partners may include, but are notlimited to, the variable region of any antibody, the target-bindingregion of a receptor, an adhesion molecule, a ligand, an enzyme, acytokine, a chemokine, or some other protein or protein domain. Smallmolecule fusion partners may include any therapeutic agent that directsthe Fc fusion to a therapeutic target. Such targets may be any molecule,preferably an extracellular receptor, which is implicated in disease.Thus, the IgG variants can be linked to one or more fusion partners.

Thus, while many embodiments herein depict antibody components such asvariable heavy and light chains or scFvs, other binding moeities can befused to Fc regions to form heterodimeric proteins. For example, asdiscussed in Kontermann, supra, any number of dual targeting strategiescan be done. For example (assuming only two binding moieties perheterodimer, e.g generally one per monomer), both monomers can bindand/or neutralize two ligands or two receptors, or bind and activate twoligands or two receptors. Similarly, one monomer may bind a receptor andthe other a ligand (again, independently activating or neutralizing thebinding partner). Further, each monomer may bind to same receptor orligand in different locations (e.g. different epitopes). See FIG. 1 ofKontermann, expressly incorporated by reference. Suitable receptors andligands are outlined below in the “Target” section.

Specific Sequences

Specific sequences for optimized CD3 binding moieties are shown in FIGS.2A-2YY.

It should be understood that the increased stability of the optimizedvariable and optimized light chains (as well as the scFv chains, listedin FIGS. 2A-2YY) can be attributed to framework regions as well as theCDRs. Thus, it should be understood that the disclosure of the entirevariable sequences (or the scFv) includes the specific disclosure of theframework regions as well, although they are not separately numbered asare the CDRs (see FIGS. 17A-17I).

FIG. 22 depicts some preferred substitutions of CDRs and frameworkregions that contribute particularly to stability. These components canbe optionally and independently combined with any other components, suchas other CDRs or frameworks. Thus, for example, the frameworksubstitutions of FIG. 22 can be combined with the following two sets ofCDRs (vhCDR1-2-3-v1CDR1-2-3):KYAMN-RIRSKYNNYATYYADSVKD-HGNFGNSYISYWAY-GSSTGAVTSGNYPN-GTKFLAP-VLWYSNRWVandTYAMN-RIRSKYNNYATYYADSVKD-HGNFGNSYVSWFAY-RSSTGAVTTSNYAN-GTKFLAP-ALWYSNLWV.

Heterodimerization Variants

Accordingly, the present invention provides heterodimeric proteins basedon the use of monomers containing variant heavy chain constant regionsas a first domain. By “monomer” herein is meant one half of theheterodimeric protein. It should be noted that antibodies are actuallytetrameric (two heavy chains and two light chains). In the context ofthe present invention, as applicable, one pair of heavy-light chains isconsidered a “monomer”. In the case where an Fv region is one fusionpartner (e.g. heavy and light chain) and a non-antibody protein isanother fusion partner, each “half” is considered a monomer.Essentially, each monomer comprises sufficient heavy chain constantregion to allow heterodimerization engineering, whether that be all theconstant region, e.g. Chl-hinge-CH2-CH3, the Fc region (CH2-CH3), orjust the CH3 domain.

The variant heavy chain constant regions can comprise all or part of theheavy chain constant region, including the full length construct,CH1-hinge-CH2-CH3, or portions thereof, including for example CH2-CH3 orCH3 alone. In addition, the heavy chain region of each monomer can bethe same backbone (CH1-hinge-CH2-CH3 or CH2-CH3) or different. N- andC-terminal truncations and additions are also included within thedefinition; for example, some pI variants include the addition ofcharged amino acids to the C-terminus of the heavy chain domain.

Furthermore, in addition to the pI substitutions outlined herein, theheavy chain regions may also contain additional amino acidsubstitutions, including changes for altering Fc binding as discussedbelow.

In addition, some monomers can utilize linkers between the variant heavychain constant region and the fusion partner. Traditional peptidelinkers can be used, including flexible linkers of glycine and serine.In some cases, the linkers for use as components of the monomer aredifferent from those defined below for the ADC constructs, and are inmany embodiments not cleavable linkers (such as those susceptible toproteases), although cleavable linkers may find use in some embodiments.

The heterodimerization variants include a number of different types ofvariants, including, but not limited to, steric variants, pI variants,and other variants (e.g. charge variants), that can be optionally andindependently combined with any other variants. In these embodiments, itis important to match “monomer A” with “monomer B”; that is, if aheterodimeric protein relies on both steric variants and pI variants,these need to be correctly matched to each monomer: e.g. the set ofsteric variants that work (1 set on monomer A, 1 set on monomer B) iscombined with pI variant sets (1 set on monomer A, 1 set on monomer B),such that the variants on each monomer are designed to achieve thedesired function.

Steric Variants

In some embodiments, the formation of heterodimers can be facilitated bythe addition of steric variants. That is, by changing amino acids ineach heavy chain, different heavy chains are more likely to associate toform the heterodimeric structure than to form homodimers with the sameFc amino acid sequences. Suitable steric variants are shown in FIGS.11A-11E.

One mechanism is generally referred to in the art as “knobs and holes”,referring to amino acid engineering that creates steric influences tofavor heterodimeric formation and disfavor homodimeric formation canalso optionally be used; this is sometimes referred to as “knobs andholes”, as described in U.S. Ser. No. 61/596,846, Ridgway et al.,Protein Engineering 9(7):617 (1996); Atwell et al., J. Mol. Biol. 1997270:26; U.S. Pat. No. 8,216,805, all of which are hereby incorporated byreference in their entirety. FIGS. 4A-4B, further described below,identifies a number of “monomer A-monomer B” pairs that rely on “knobsand holes”. In addition, as described in Merchant et al., NatureBiotech. 16:677 (1998), these “knobs and hole” mutations can be combinedwith disulfide bonds to skew formation to heterodimerization. Some ofthese variants are shown in the Figures.

An additional mechanism that finds use in the generation of heterodimersis sometimes referred to as “electrostatic steering” as described inGunasekaran et al., J. Biol. Chem. 285(25):19637 (2010), herebyincorporated by reference in its entirety. This is sometimes referred toherein as “charge pairs”. In this embodiment, electrostatics are used toskew the formation towards heterodimerization. As those in the art willappreciate, these may also have an effect on pI, and thus onpurification, and thus could in some cases also be considered pIvariants. However, as these were generated to force heterodimerizationand were not used as purification tools, they are classified as “stericvariants”. These include, but are not limited to, D221E/P228E/L368Epaired with D221R/P228R/K409R (e.g. these are “monomer correspondingsets) and C220E/P228E/368E paired with C220R/E224R/P228R/K409R.

Additional monomer A and monomer B variants that can be combined withother variants, optionally and independently in any amount, such as pIvariants outlined herein or other steric variants that are shown in FIG.37 of US 2012/0149876, the figure and legend of which are incorporatedexpressly by reference herein.

In some embodiments, the steric variants outlined herein can beoptionally and independently incorporated with any pI variant (or othervariants such as Fc variants, FcRn variants, etc.) into one or bothmonomers.

pI (Isoelectric point) Variants for Heterodimers

In general, as will be appreciated by those in the art, there are twogeneral categories of pI variants: those that increase the pI of theprotein (basic changes) and those that decrease the pI of the protein(acidic changes). As described herein, all combinations of thesevariants can be done: one monomer may be wild type, or a variant thatdoes not display a significantly different pI from wild-type, and theother can be either more basic or more acidic. Alternatively, eachmonomer is changed, one to more basic and one to more acidic.

Preferred combinations of pI variants are shown in FIG. 82.

Heavy Chain Acidic pI Changes

Accordingly, when one monomer comprising a variant heavy chain constantdomain is to be made more positive (e.g. lower the pI), one or more ofthe following substitutions can be made: S119E, K133E, K133Q, T164E,K205E, K205Q, N208D, K210E, K210Q, K274E, K320E, K322E, K326E, K334E,R355E, K392E, a deletion of K447, adding peptide DEDE at the c-terminus,G137E, N203D, K274Q, R355Q, K392N and Q419E. As outlined herein andshown in the figures, these changes are shown relative to IgG1, but allisotypes can be altered this way, as well as isotype hybrids.

In the case where the heavy chain constant domain is from IgG2-4, R133Eand R133Q can also be used.

Basic pI changes

Accordingly, when one monomer comprising a variant heavy chain constantdomain is to be made more negative (e.g. increase the pI), one or moreof the following substitutions can be made: Q196K, P217R, P228R, N276Kand H435R. As outlined herein and shown in the figures, these changesare shown relative to IgG1, but all isotypes can be altered this way, aswell as isotype hybrids.

Antibody Heterodimers Light Chain Variants

In the case of antibody based heterodimers, e.g. where at least one ofthe monomers comprises a light chain in addition to the heavy chaindomain, pI variants can also be made in the light chain. Amino acidsubstitutions for lowering the pI of the light chain include, but arenot limited to, K126E, K126Q, K145E, K145Q, N152D, S156E, K169E, S202E,K207E and adding peptide DEDE at the c-terminus of the light chain.Changes in this category based on the constant lambda light chaininclude one or more substitutions at R108Q, Q124E, K126Q, N138D, K145Tand Q199E. In addition, increasing the pI of the light chains can alsobe done.

Isotypic Variants

In addition, many embodiments of the invention rely on the “importation”of pI amino acids at particular positions from one IgG isotype intoanother, thus reducing or eliminating the possibility of unwantedimmunogenicity being introduced into the variants. That is, IgG1 is acommon isotype for therapeutic antibodies for a variety of reasons,including high effector function. However, the heavy constant region ofIgG1 has a higher pI than that of IgG2 (8.10 versus 7.31). Byintroducing IgG2 residues at particular positions into the IgG1backbone, the pI of the resulting monomer is lowered (or increased) andadditionally exhibits longer serum half-life. For example, IgG1 has aglycine (pI 5.97) at position 137, and IgG2 has a glutamic acid (pI3.22); importing the glutamic acid will affect the pI of the resultingprotein. As is described below, a number of amino acid substitutions aregenerally required to significant affect the pI of the variant antibody.However, it should be noted as discussed below that even changes in IgG2molecules allow for increased serum half-life.

In other embodiments, non-isotypic amino acid changes are made, eitherto reduce the overall charge state of the resulting protein (e.g. bychanging a higher pI amino acid to a lower pI amino acid), or to allowaccommodations in structure for stability, etc. as is more furtherdescribed below.

In addition, by pI engineering both the heavy and light constantdomains, significant changes in each monomer of the heterodimer can beseen. As discussed herein, having the pIs of the two monomers differ byat least 0.5 can allow separation by ion exchange chromatography orisoelectric focusing, or other methods sensitive to isoelectric point.

Calculating pI

The pI of each monomer can depend on the pI of the variant heavy chainconstant domain and the pI of the total monomer, including the variantheavy chain constant domain and the fusion partner. Thus, in someembodiments, the change in pI is calculated on the basis of the variantheavy chain constant domain, using the chart in the Figures.Alternatively, the pI of each monomer can be compared.

pI Variants that Also Confer Better FcRn In Vitro Binding

In the case where the pI variant decreases the pI of the monomer, theycan have the added benefit of improving serum retention in vivo.

Although still under examination, Fc regions are believed to have longerhalf-lives in vivo, because binding to FcRn at pH 6 in an endosomesequesters the Fc (Ghetie and Ward, 1997 Immunol Today. 18(12): 592-598,entirely incorporated by reference). The endosomal compartment thenrecycles the Fc to the cell surface. Once the compartment opens to theextracellular space, the higher pH, ˜7.4, induces the release of Fc backinto the blood. In mice, Dall' Acqua et al. showed that Fc mutants withincreased FcRn binding at pH 6 and pH 7.4 actually had reduced serumconcentrations and the same half life as wild-type Fc (Dall' Acqua etal. 2002, J. Immunol. 169:5171-5180, entirely incorporated byreference). The increased affinity of Fc for FcRn at pH 7.4 is thoughtto forbid the release of the Fc back into the blood. Therefore, the Fcmutations that will increase Fc's half-life in vivo will ideallyincrease FcRn binding at the lower pH while still allowing release of Fcat higher pH. The amino acid histidine changes its charge state in thepH range of 6.0 to 7.4. Therefore, it is not surprising to find Hisresidues at important positions in the Fc/FcRn complex.

Recently it has been suggested that antibodies with variable regionsthat have lower isoelectric points may also have longer serum half-lives(Igawa et al., 2010 PEDS. 23(5): 385-392, entirely incorporated byreference). However, the mechanism of this is still poorly understood.Moreover, variable regions differ from antibody to antibody. Constantregion variants with reduced pI and extended half-life would provide amore modular approach to improving the pharmacokinetic properties ofantibodies, as described herein.

Combination of Heterodimeric Variants

As will be appreciated by those in the art, all of the recitedheterodimerization variants can be optionally and independently combinedin any way, as long as they retain their “strandedness” or “monomerpartition”. In addition, all of these variants can be combined into anyof the hterodimerization formats. See FIGS. 21A-21B.

In the case of pI variants, while embodiments finding particular use areshown in the Figures, other combinations can be generated, following thebasic rule of altering the pI difference between two monomers tofacilitate purification.

Suitable Multispecific Formats

As will be appreciated by those in the art, there are a wide variety ofpossible multispecific formats that find use in the present invention,see for example Kontermann, mAbs 4(2):182-197 (2012), herebyincorporated by reference in its entirety and particularly Tables 1 and2 and FIGS. 1 and 2, with specific reference to the constructs ofKontermann that contain an Fc region. See also Klein et al., Of use inthe present invention are heterodimers that contain constant heavy chainand/or constant light chain regions, and in particular, Fc domains. Thatis, some variants discussed herein are within the vhCH1, although manyof the variants are within the Fc domain (hinge-CH2-CH3).

As will be appreciated by those in the art and discussed more fullybelow, the heterodimeric fusion proteins of the present invention cantake on a wide variety of configurations, as are generally depicted inthe Figures. Some figures depict “single ended” configurations, wherethere is one type of specificity on one “arm” of the molecule and adifferent specificity on the other “arm”. Other figures depict “dualended” configurations, where there is at least one type of specificityat the “top” of the molecule and one or more different specificities atthe “bottom” of the molecule. Furthermore as is shown, these twoconfigurations can be combined, where there can be triple or quadruplespecificities based on the particular combination. Thus, the presentinvention provides “multispecific” binding proteins, includingmultispecific antibodies.

In some embodiments, the heterodimers resemble traditional antibodiesalthough they are bispecific and have two different variable regions;see FIG. 78. As outlined herein, the constant regions compriseheterodimerization variants, such as steric variants (“knobs in holes”,sometimes referred to in the art as “kih” variants) or pI variants, etc.In some cases, to reduce the complexity with regard to the light chains,some of these formats utilize variable regions that share a common lightchain (e.g. two separate heavy chains with a light chain that willassemble with both but confers two different specificities).

In some embodiments, the heterodimers are bispecific in a formatgenerally referred to in the art as “CrossMab”. In this embodiment, inaddition to using the heterodimeric variants described herein, one heavychain monomer and one light chain monomer are also engineered such thatthe heavy chain monomer comprises a constant light region in place ofthe vhCH1domain, and the light chain contains the vhCH1 region with thevariable light region. This ensures that the correct light chains willpair with the correct heavy chains. See FIGS. 7A-7U and Schaefer et al.,PNAS 108(27) 11187-11192 (hereby incorporated by reference in itsentirety.

In some embodiments, sometimes referred to in the art as IgG-scFab, oneof the heavy chains has a scFab on it, such that one antigen is engagedbivalently and the other monovalently (e.g. two binding regions on one“end” and a single binding region on the other “end”). See FIGS. 7A-7U.

In some embodiments, sometimes referred to as mAb-Fv, each heavy chainof the heterodimer has an additional variable region on the terminus Onemonomer has the variable heavy domain and the other monomer has avariable light domain, see FIGS. 7A-7U. See for example PCTUS2010/047741, hereby incorporated by reference. In this embodiment, ingeneral, there are two different types of antibody analogs that allowfor co-engagement mechanisms, one that utilizes three antigen bindingdomains (e.g. one antigen is bound bivalently and the other is boundmonovalently, although as is further described below, there can also bethree different antigens that are bound or a single antigen), and onethat relies on two antigen binding domains (e.g. each antigen is boundmonovalently).

Additional Modifications

In addition to the modifications outlined above, other modifications canbe made. For example, the molecules may be stabilized by theincorporation of disulphide bridges linking the VH and VL domains(Reiter et al., 1996, Nature Biotech. 14:1239-1245, entirelyincorporated by reference). In addition, there are a variety of covalentmodifications of antibodies that can be made as outlined below.

Covalent modifications of antibodies are included within the scope ofthis invention, and are generally, but not always, donepost-translationally. For example, several types of covalentmodifications of the antibody are introduced into the molecule byreacting specific amino acid residues of the antibody with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C-terminal residues.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesmay also be derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole and the like.

In addition, modifications at cysteines are particularly useful inantibody-drug conjugate (ADC) applications, further described below. Insome embodiments, the constant region of the antibodies can beengineered to contain one or more cysteines that are particularly “thiolreactive”, so as to allow more specific and controlled placement of thedrug moiety. See for example U.S. Pat. No. 7,521,541, incorporated byreference in its entirety herein.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing alpha-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pKa of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using 1251 or 1311 to prepare labeled proteinsfor use in radioimmunoassay, the chloramine T method described abovebeing suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R′—N═C═N—R′), where R and R′ are optionallydifferent alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Derivatization with bifunctional agents is useful for crosslinkingantibodies to a water-insoluble support matrix or surface for use in avariety of methods, in addition to methods described below. Commonlyused crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such ascynomolgusogen bromide-activated carbohydrates and the reactivesubstrates described in U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128;4,247,642; 4,229,537; and 4,330,440, all entirely incorporated byreference, are employed for protein immobilization.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively.Alternatively, these residues are deamidated under mildly acidicconditions. Either form of these residues falls within the scope of thisinvention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, pp. 79-86 [1983],entirely incorporated by reference), acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

In addition, as will be appreciated by those in the art, labels(including fluorescent, enzymatic, magnetic, radioactive, etc. can allbe added to the antibodies (as well as the other compositions of theinvention).

Glycosylation

Another type of covalent modification is alterations in glycosylation.In another embodiment, the antibodies disclosed herein can be modifiedto include one or more engineered glycoforms. By “engineered glycoform”as used herein is meant a carbohydrate composition that is covalentlyattached to the antibody, wherein said carbohydrate composition differschemically from that of a parent antibody. Engineered glycoforms may beuseful for a variety of purposes, including but not limited to enhancingor reducing effector function. A preferred form of engineered glycoformis afucosylation, which has been shown to be correlated to an increasein ADCC function, presumably through tighter binding to the FcγRIIIareceptor. In this context, “afucosylation” means that the majority ofthe antibody produced in the host cells is substantially devoid offucose, e.g. 90-95-98% of the generated antibodies do not haveappreciable fucose as a component of the carbohydrate moiety of theantibody (generally attached at N297 in the Fc region). Definedfunctionally, afucosylated antibodies generally exhibit at least a 50%or higher affinity to the FcγRIIIa receptor.

Engineered glycoforms may be generated by a variety of methods known inthe art (Umaña et al., 1999, Nat Biotechnol 17:176-180; Davies et al.,2001, Biotechnol Bioeng 74:288-294; Shields et al., 2002, J Biol Chem277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473; U.S.Pat. No. 6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser. No. 10/113,929;PCT WO 00/61739A1; PCT WO 01/29246A1; PCT WO 02/31140A1; PCT WO02/30954A1, all entirely incorporated by reference; (Potelligent®technology [Biowa, Inc., Princeton, N.J.]; GlycoMAb® glycosylationengineering technology [Glycart Biotechnology AG, Zurich, Switzerland]).Many of these techniques are based on controlling the level offucosylated and/or bisecting oligosaccharides that are covalentlyattached to the Fc region, for example by expressing an IgG in variousorganisms or cell lines, engineered or otherwise (for example Lec-13 CHOcells or rat hybridoma YB2/0 cells, by regulating enzymes involved inthe glycosylation pathway (for example FUT8 [α1,6-fucosyltranserase]and/or β1-4-N-acetylglucosaminyltransferase III [GnTIII]), or bymodifying carbohydrate(s) after the IgG has been expressed. For example,the “sugar engineered antibody” or “SEA technology” of Seattle Geneticsfunctions by adding modified saccharides that inhibit fucosylationduring production; see for example 20090317869, hereby incorporated byreference in its entirety. Engineered glycoform typically refers to thedifferent carbohydrate or oligosaccharide; thus an antibody can includean engineered glycoform.

Alternatively, engineered glycoform may refer to the IgG variant thatcomprises the different carbohydrate or oligosaccharide. As is known inthe art, glycosylation patterns can depend on both the sequence of theprotein (e.g., the presence or absence of particular glycosylation aminoacid residues, discussed below), or the host cell or organism in whichthe protein is produced. Particular expression systems are discussedbelow.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tri-peptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tri-peptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid,most commonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tri-peptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thestarting sequence (for O-linked glycosylation sites). For ease, theantibody amino acid sequence is preferably altered through changes atthe DNA level, particularly by mutating the DNA encoding the targetpolypeptide at preselected bases such that codons are generated thatwill translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on theantibody is by chemical or enzymatic coupling of glycosides to theprotein. These procedures are advantageous in that they do not requireproduction of the protein in a host cell that has glycosylationcapabilities for N- and O-linked glycosylation. Depending on thecoupling mode used, the sugar(s) may be attached to (a) arginine andhistidine, (b) free carboxyl groups, (c) free sulfhydryl groups such asthose of cysteine, (d) free hydroxyl groups such as those of serine,threonine, or hydroxyproline, (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan, or (f) the amide group ofglutamine. These methods are described in WO 87/05330 and in Aplin andWriston, 1981, CRC Crit. Rev. Biochem., pp. 259-306, both entirelyincorporated by reference.

Removal of carbohydrate moieties present on the starting antibody (e.g.post-translationally) may be accomplished chemically or enzymatically.Chemical deglycosylation requires exposure of the protein to thecompound trifluoromethanesulfonic acid, or an equivalent compound. Thistreatment results in the cleavage of most or all sugars except thelinking sugar (N-acetylglucosamine or N-acetylgalactosamine), whileleaving the polypeptide intact. Chemical deglycosylation is described byHakimuddin et al., 1987, Arch. Biochem. Biophys. 259:52 and by Edge etal., 1981, Anal. Biochem. 118:131, both entirely incorporated byreference. Enzymatic cleavage of carbohydrate moieties on polypeptidescan be achieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al., 1987, Meth. Enzymol. 138:350, entirelyincorporated by reference. Glycosylation at potential glycosylationsites may be prevented by the use of the compound tunicamycin asdescribed by Duskin et al., 1982, J. Biol. Chem. 257:3105, entirelyincorporated by reference. Tunicamycin blocks the formation ofprotein-N-glycoside linkages.

Another type of covalent modification of the antibody comprises linkingthe antibody to various nonproteinaceous polymers, including, but notlimited to, various polyols such as polyethylene glycol, polypropyleneglycol or polyoxyalkylenes, in the manner set forth in, for example,2005-2006 PEG Catalog from Nektar Therapeutics (available at the Nektarwebsite) U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417;4,791,192 or 4,179,337, all entirely incorporated by reference. Inaddition, as is known in the art, amino acid substitutions may be madein various positions within the antibody to facilitate the addition ofpolymers such as PEG. See for example, U.S. Publication No.2005/0114037A1, entirely incorporated by reference.

Other Fc Modifications

In addition to heterodimerization variants, other amino acidmodifications (particularly amino acid substitutions) find use to alteradditional properties of the heterodimer.

FcγR Variants

In one embodiment, the heterodimers of the invention can include aminoacid modifications to alter binding to one or more of the FcγRreceptors. Substitutions that result in increased binding as well asdecreased binding can be useful. For example, it is known that increasedbinding to FcγRIIIa generally results in increased ADCC (antibodydependent cell-mediated cytotoxicity; the cell-mediated reaction whereinnonspecific cytotoxic cells that express FcγRs recognize bound antibodyon a target cell and subsequently cause lysis of the target cell).Similarly, decreased binding to FcγRIIb (an inhibitory receptor) can bebeneficial as well in some circumstances. Amino acid substitutions thatfind use in the present invention include those listed in U.S. Ser. No.11/124,620 (particularly FIG. 41, specifically incorporated herein),Ser. No. 11/174,287, Ser. No. 11/396,495, Ser. No. 11/538,406, all ofwhich are expressly incorporated herein by reference in their entiretyand specifically for the variants disclosed therein.

Particular variants that find use include, but are not limited to, 236A,239D, 239E, 332E, 332D, 239D/332E, 267D, 267E, 328F, 267E/328F,236A/332E, 239D/332E/330Y, 239D, 332E/330L, 243L, 236R, 328R, 236R/328R,298A and 299T. Additional suitable Fc variants are found in FIG. 41 ofUS 2006/0024298, the figure and legend of which are hereby incorporatedby reference in their entirety.

FcRn Modifications

In addition, there are additional Fc substitutions that find use inincreased binding to the FcRn receptor and/or increased serum half life,as specifically disclosed in U.S. Ser. No. 12/341,769, herebyincorporated by reference in its entirety (particularly FIGS. 9A-9C andFIGS. 10A-10E), including, but not limited to, 434S, 434A, 428L, 308F,259I, 428L/434S, 259I/308F, 436I/428L, 436I or V/434S, 436V/428L, 252Y,252Y/254T/256E and 259I/308F/428L.

Binding Moieties/Targets

The heterodimeric proteins (for example the heterodimericimmunoglobulins) of the invention include at least a first bindingmoiety that is an anti-CD3 Fv region (including, as outlined herein,scFv variable regions). As described herein, additional binding moieties(either antibody components such as other Fvs, other scFvs, Fabs, etc.)may target virtually any antigens. As noted above, there are a widevariety of suitable heterodimeric antibody formats, with some preferablyco-engage two target antigens, although in some cases, three or fourantigens can be engaged.

Combinations that find particular use in a variety of embodimentsinclude heterodimers that bind two antigens: CD3 and an antigen selectedfrom the group consisting of CD5, CD20, CD30, CD33, CD38, CD40, EGFR,EpCAM, Her2 and HM1.24.

Particular suitable applications of the immunoglobulins herein areco-target pairs for which it is beneficial or critical to engage eachtarget antigen monovalently. Such antigens may be, for example, immunereceptors that are activated upon immune complexation. Cellularactivation of many immune receptors occurs only by cross-linking,achieved typically by antibody/antigen immune complexes, or via effectorcell to target cell engagement. For some immune receptors, for examplethe CD3 signaling receptor on T cells, activation only upon engagementwith co-engaged target is critical, as nonspecific cross-linking in aclinical setting can elicit a cytokine storm and toxicity.Therapeutically, by engaging such antigens monovalently rather thanmultivalently, using the immunoglobulins herein, such activation occursonly in response to cross-linking only in the microenvironment of theprimary target antigen. The ability to target two different antigenswith different valencies is a novel and useful aspect of the presentinvention. Examples of target antigens for which it may betherapeutically beneficial or necessary to co-engage monovalentlyinclude but are not limited to immune activating receptors such as CD3,FcγRs, toll-like receptors (TLRs) such as TLR4 and TLR9, cytokine,chemokine, cytokine receptors, and chemokine receptors.

Virtually any antigen may be targeted by the immunoglobulins herein,including but not limited to proteins, subunits, domains, motifs, and/orepitopes belonging to the following list of target antigens, whichincludes both soluble factors such as cytokines and membrane-boundfactors, including transmembrane receptors: 17-IA, 4-1BB, 4Dc,6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 Adenosine Receptor, A33, ACE,ACE-2, Activin, Activin A, Activin AB, Activin B, Activin C, ActivinRIA, Activin RIA ALK-2, Activin RIB ALK-4, Activin RIIA, Activin RIIB,ADAM, ADAM10, ADAM12, ADAM15, ADAM17/TACE, ADAM8, ADAM9, ADAMTS,ADAMTS4, ADAMTS5, Addressins, aFGF, ALCAM, ALK, ALK-1, ALK-7,alpha-1-antitrypsin, alpha-V/beta-1 antagonist, ANG, Ang, APAF-1, APE,APJ, APP, APRIL, AR, ARC, ART, Artemin, anti-Id, ASPARTIC, Atrialnatriuretic factor, av/b3 integrin, Ax1, b2M, B7-1, B7-2, B7-H,B-lymphocyte Stimulator (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1,BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM,BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 Osteogenin, BMP-4 BMP-2b,BMP-5, BMP-6 Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA(ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMPs, b-NGF,BOK, Bombesin, Bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC,complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8,Calcitonin, cAMP, carcinoembryonic antigen (CEA), carcinoma-associatedantigen, Cathepsin A, Cathepsin B, Cathepsin C/DPPI, Cathepsin D,Cathepsin E, Cathepsin H, Cathepsin L, Cathepsin 0, Cathepsin S,Cathepsin V, Cathepsin X/Z/P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12,CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21,CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6,CCL7, CCL8, CCL9/10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5,CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8,CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20,CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67proteins), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54,CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123,CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR,cGMP, CINC, Clostridium botulinum toxin, Clostridium perfringens toxin,CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK,CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5,CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14,CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6,cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, Decayaccelerating factor, des(1-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1,Dnase, Dpp, DPPIV/CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR(ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, Enkephalinase, eNOS,Eot, eotaxin1, EpCAM, Ephrin B2/EphB4, EPO, ERCC, E-selectin, ET-1,Factor Ha, Factor VII, Factor VIIIc, Factor IX, fibroblast activationprotein (FAP), Fas, FcR1, FEN-1, Ferritin, FGF, FGF-19, FGF-2, FGF3,FGF-8, FGFR, FGFR-3, Fibrin, FL, FLIP, Flt-3, Flt-4, Folliclestimulating hormone, Fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6,FZD7, FZD8, FZD9, FZD10, G250, Gas 6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1,GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7(BMP-12, CDMP-3), GDF-8 (Myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF,GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, Glucagon, Glut4, glycoprotein IIb/IIIa (GP IIb/IIIa), GM-CSF, gp130, gp72, GRO, Growthhormone releasing factor, Hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMVgB envelope glycoprotein, HCMV) gH envelope glycoprotein, HCMV UL,Hemopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2/neu(ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gBglycoprotein, HSV gD glycoprotein, HGFA, High molecular weightmelanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp 120 V3loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin,human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, 1-309,IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF,IGF binding proteins, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R,IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10,IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (INF)-alpha,INF-beta, INF-gamma, Inhibin, iNOS, Insulin A-chain, Insulin B-chain,Insulin-like growth factor 1, integrin alpha2, integrin alpha3, integrinalpha4, integrin alpha4/beta1, integrin alpha4/beta7, integrin alpha5(alphaV), integrin alpha5/beta1, integrin alpha5/beta3, integrin alpha6,integrin beta1, integrin beta2, interferon gamma, IP-10, I-TAC, JE,Kallikrein 2, Kallikrein 5, Kallikrein 6, Kallikrein 11, Kallikrein 12,Kallikrein 14, Kallikrein 15, Kallikrein L1, Kallikrein L2, KallikreinL3, Kallikrein L4, KC, KDR, Keratinocyte Growth Factor (KGF), laminin 5,LAMP, LAP, LAP (TGF-1), Latent TGF-1, Latent TGF-1 bp1, LBP, LDGF,LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3,Lfo, LIF, LIGHT, lipoproteins, LIX, LKN, Lptn, L-Selectin, LT-a, LT-b,LTB4, LTBP-1, Lung surfactant, Luteinizing hormone, Lymphotoxin BetaReceptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF,MDC, Mer, METALLOPROTEASES, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG,MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13,MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo,MSK, MSP, mucin (Muc1), MUC18, Muellerian-inhibitin substance, Mug,MuSK, NAIP, NAP, NCAD, N-Cadherin, NCA 90, NCAM, NCAM, Neprilysin,Neurotrophin-3, -4, or -6, Neurturin, Neuronal growth factor (NGF),NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN,OSM, OX40L, OX40R, p150, p95, PADPr, Parathyroid hormone, PARC, PARP,PBR, PBSF, PCAD, P-Cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4,PGE, PGF, PGI2, PGD2, PIN, PLA2, placental alkaline phosphatase (PLAP),P1GF, PLP, PP14, Proinsulin, Prorelaxin, Protein C, PS, PSA, PSCA,prostate specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51,RANK, RANKL, RANTES, RANTES, Relaxin A-chain, Relaxin B-chain, renin,respiratory syncytial virus (RSV) F, RSV Fgp, Ret, Rheumatoid factors,RLIP76, RPA2, RSK, 5100, SCF/KL, SDF-1, SERINE, Serum albumin, sFRP-3,Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat,STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72),TARC, TCA-3, T-cell receptors (e.g., T-cell receptor alpha/beta), TdT,TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkalinephosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan Specific,TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta RIM, TGF-beta RIII,TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, Thrombin, ThymusCk-1, Thyroid stimulating hormone, Tie, TIMP, TIQ, Tissue Factor,TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha beta, TNF-beta2, TNFc,TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSF10B (TRAIL R2DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID),TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R),TNFRSF11B (OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B (TACI),TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF 16(NGFR p75NTR), TNFRSF 17 (BCMA), TNFRSF 18 (GITR AITR), TNFRSF19 (TROYTAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF RI CD120a, p55-60),TNFRSF1B (TNF RII CD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNFRIII, TNFC R), TNFRSF4 (OX40 ACT35, TXGP1R), TNFRSF5 (CD40 p50), TNFRSF6(Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3 M68, TR6), TNFRSF7 (CD27),TNFRSF8 (CD30), TNFRSF9 (4-1BB CD137, ILA), TNFRSF21 (DR6), TNFRSF22(DcTRAIL R2 TNFRH2), TNFRST23 (DcTRAIL R1 TNFRH1), TNFRSF25 (DR3 Apo-3,LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 Ligand, TL2), TNFSF11(TRANCE/RANK Ligand ODF, OPG Ligand), TNFSF12 (TWEAK Apo-3 Ligand, DR3Ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK,TNFSF20), TNFSF14 (LIGHT HVEM Ligand, LTg), TNFSF15 (TL1A/VEGI), TNFSF18(GITR Ligand AITR Ligand, TL6), TNFSF1A (TNF-a Conectin, DIF, TNFSF2),TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 Ligandgp34, TXGP1), TNFSF5 (CD40 Ligand CD154, gp39, HIGM1, IMD3, TRAP),TNFSF6 (Fas Ligand Apo-1 Ligand, APT1 Ligand), TNFSF7 (CD27 LigandCD70), TNFSF8 (CD30 Ligand CD153), TNFSF9 (4-1BB Ligand CD137 Ligand),TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE,transferring receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associatedantigen CA 125, tumor-associated antigen expressing Lewis Y relatedcarbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, Urokinase, VCAM, VCAM-1,VECAD, VE-Cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3(fit-4), VEGI, VIM, Viral antigens, VLA, VLA-1, VLA-4, VNR integrin, vonWillebrands factor, WIF-1, WNT1, WNT2, WNT2B/13, WNT3, WNT3A, WNT4,WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B,WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD,and receptors for hormones and growth factors. To form the bispecific ortrispecific antibodies of the invention, antibodies to any combinationof these antigens can be made; that is, each of these antigens can beoptionally and independently included or excluded from a multispecificantibody according to the present invention.

Exemplary antigens that may be targeted specifically by theimmunoglobulins of the invention include but are not limited to: CD20,CD19, Her2, EGFR, EpCAM, CD3, FcγRIIIa (CD16), FcγRIIa (CD32a), FcγRIIb(CD32b), FcγRI (CD64), Toll-like receptors (TLRs) such as TLR4 and TLR9,cytokines such as IL-2, IL-5, IL-13, IL-12, IL-23, and TNFα, cytokinereceptors such as IL-2R, chemokines, chemokine receptors, growth factorssuch as VEGF and HGF, and the like. To form the bispecific ortrispecific antibodies of the invention, antibodies to any combinationof these antigens can be made; that is, each of these antigens can beoptionally and independently included or excluded from a multispecificantibody according to the present invention.

The choice of suitable target antigens and co-targets depends on thedesired therapeutic application. Some targets that have provenespecially amenable to antibody therapy are those with signalingfunctions. Other therapeutic antibodies exert their effects by blockingsignaling of the receptor by inhibiting the binding between a receptorand its cognate ligand. Another mechanism of action of therapeuticantibodies is to cause receptor down regulation. Other antibodies do notwork by signaling through their target antigen. The choice of co-targetswill depend on the detailed biology underlying the pathology of theindication that is being treated.

Monoclonal antibody therapy has emerged as an important therapeuticmodality for cancer (Weiner et al., 2010, Nature Reviews Immunology10:317-327; Reichert et al., 2005, Nature Biotechnology 23[9]:1073-1078;herein expressly incorporated by reference). For anti-cancer treatmentit may be desirable to target one antigen (antigen-1) whose expressionis restricted to the cancerous cells while co-targeting a second antigen(antigen-2) that mediates some immunulogical killing activity. For othertreatments it may be beneficial to co-target two antigens, for exampletwo angiogenic factors or two growth factors, that are each known toplay some role in proliferation of the tumor. Exemplary co-targets foroncology include but are not limited to HGF and VEGF, IGF-1R and VEGF,Her2 and VEGF, CD19 and CD3, CD20 and CD3, Her2 and CD3, CD19 andFcγRIIIa, CD20 and FcγRIIIa, Her2 and FcγRIIIa. An immunoglobulin of theinvention may be capable of binding VEGF and phosphatidylserine; VEGFand ErbB3; VEGF and PLGF; VEGF and ROBO4; VEGF and BSG2; VEGF and CDCP1;VEGF and ANPEP; VEGF and c-MET; HER-2 and ERB3; HER-2 and BSG2; HER-2and CDCP1; HER-2 and ANPEP; EGFR and CD64; EGFR and BSG2; EGFR andCDCP1; EGFR and ANPEP; IGF1R and PDGFR; IGF1R and VEGF; IGF1R and CD20;CD20 and CD74; CD20 and CD30; CD20 and DR4; CD20 and VEGFR2; CD20 andCD52; CD20 and CD4; HGF and c-MET; HGF and NRP1; HGF andphosphatidylserine; ErbB3 and IGF1R; ErbB3 and IGF1,2; c-Met and Her-2;c-Met and NRP1; c-Met and IGF1R; IGF1,2 and PDGFR; IGF1,2 and CD20;IGF1,2 and IGF1R; IGF2 and EGFR; IGF2 and HER2; IGF2 and CD20; IGF2 andVEGF; IGF2 and IGF1R; IGF1 and IGF2; PDGFRa and VEGFR2; PDGFRa and PLGF;PDGFRa and VEGF; PDGFRa and c-Met; PDGFRa and EGFR; PDGFRb and VEGFR2;PDGFRb and c-Met; PDGFRb and EGFR; RON and c-Met; RON and MTSP1; RON andMSP; RON and CDCP1; VGFR1 and PLGF; VGFR1 and RON; VGFR1 and EGFR;VEGFR2 and PLGF; VEGFR2 and NRP1; VEGFR2 and RON; VEGFR2 and DLL4;VEGFR2 and EGFR; VEGFR2 and ROBO4; VEGFR2 and CD55; LPA and S1P; EPHB2and RON; CTLA4 and VEGF; CD3 and EPCAM; CD40 and IL6; CD40 and IGF; CD40and CD56; CD40 and CD70; CD40 and VEGFR1; CD40 and DR5; CD40 and DR4;CD40 and APRIL; CD40 and BCMA; CD40 and RANKL; CD28 and MAPG; CD80 andCD40; CD80 and CD30; CD80 and CD33; CD80 and CD74; CD80 and CD2; CD80and CD3; CD80 and CD19; CD80 and CD4; CD80 and CD52; CD80 and VEGF; CD80and DR5; CD80 and VEGFR2; CD22 and CD20; CD22 and CD80; CD22 and CD40;CD22 and CD23; CD22 and CD33; CD22 and CD74; CD22 and CD19; CD22 andDR5; CD22 and DR4; CD22 and VEGF; CD22 and CD52; CD30 and CD20; CD30 andCD22; CD30 and CD23; CD30 and CD40; CD30 and VEGF; CD30 and CD74; CD30and CD19; CD30 and DR5; CD30 and DR4; CD30 and VEGFR2; CD30 and CD52;CD30 and CD4; CD138 and RANKL; CD33 and FTL3; CD33 and VEGF; CD33 andVEGFR2; CD33 and CD44; CD33 and DR4; CD33 and DR5; DR4 and CD137; DR4and IGF1,2; DR4 and IGF1R; DR4 and DR5; DR5 and CD40; DR5 and CD137; DR5and CD20; DR5 and EGFR; DR5 and IGF1,2; DR5 and IGFR, DR5 and HER-2, andEGFR and DLL4. Other target combinations include one or more members ofthe EGF/erb-2/erb-3 family.

Other targets (one or more) involved in oncological diseases that theimmunoglobulins herein may bind include, but are not limited to thoseselected from the group consisting of: CD52, CD20, CD19, CD3, CD4, CD8,BMP6, IL12A, ILIA, IL1B, IL2, IL24, INHA, TNF, TNFSF10, BMP6, EGF, FGF1,FGF10, FGF11, FGF12, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF2,FGF20, FGF21, FGF22, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9,GRP, IGF1, IGF2, IL12A, IL1A, IL1B, IL2, INHA, TGFA, TGFB1, TGFB2,TGFB3, VEGF, CDK2, FGF10, FGF18, FGF2, FGF4, FGF7, IGF1R, IL2, BCL2,CD164, CDKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2B, CDKN2C, CDKN3, GNRH1,IGFBP6, IL1A, IL1B, ODZ1, PAWR, PLG, TGFB1I1, AR, BRCA1, CDK3, CDK4,CDK5, CDK6, CDK7, CDK9, E2F1, EGFR, ENO1, ERBB2, ESR1, ESR2, IGFBP3,IGFBP6, IL2, INSL4, MYC, NOX5, NR6A1, PAP, PCNA, PRKCQ, PRKD1, PRL,TP53, FGF22, FGF23, FGF9, IGFBP3, IL2, INHA, KLK6, TP53, CHGB, GNRH1,IGF1, IGF2, INHA, INSL3, INSL4, PRL, KLK6, SHBG, NR1D1, NR1H3, NR1I3,NR2F6, NR4A3, ESR1, ESR2, NROB1, NROB2, NR1D2, NR1H2, NR1H4, NR112,NR2C1, NR2C2, NR2E1, NR2E3, NR2F1, NR2F2, NR3C1, NR3C2, NR4A1, NR4A2,NR5A1, NR5A2, NR6 μl, PGR, RARB, FGF1, FGF2, FGF6, KLK3, KRT1, APOC1,BRCA1, CHGA, CHGB, CLU, COL1A1, COL6A1, EGF, ERBB2, ERK8, FGF1, FGF10,FGF11, FGF13, FGF14, FGF16, FGF17, FGF18, FGF2, FGF20, FGF21, FGF22,FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, GNRH1, IGF1, IGF2,IGFBP3, IGFBP6, IL12A, IL1A, IL1B, IL2, IL24, INHA, INSL3, INSL4, KLK10,KLK12, KLK13, KLK14, KLK15, KLK3, KLK4, KLK5, KLK6, KLK9, MMP2, MMP9,MSMB, NTN4, ODZ1, PAP, PLAU, PRL, PSAP, SERPINA3, SHBG, TGFA, TIMP3,CD44, CDH1, CDH10, CDH19, CDH20, CDH7, CDH9, CDH1, CDH10, CDH13, CDH18,CDH19, CDH20, CDH7, CDH8, CDH9, ROBO2, CD44, ILK, ITGA1, APC, CD164,COL6A1, MTSS1, PAP, TGFB1I1, AGR2, AIG1, AKAP1, AKAP2, CANT1, CAV1,CDH12, CLDN3, CLN3, CYB5, CYC1, DAB21P, DES, DNCL1, ELAC2, ENO2, ENO3,FASN, FLJ12584, FLJ25530, GAGEB1, GAGEC1, GGT1, GSTP1, HIP 1, HUMCYT2A,IL29, K6HF, KAI1, KRT2A, MIB1, PART1, PATE, PCA3, PIAS2, PIK3CG, PPID,PR1, PSCA, SLC2A2, SLC33 μl, SLC43 μl, STEAP, STEAP2, TPM1, TPM2, TRPC6,ANGPT1, ANGPT2, ANPEP, ECGF1, EREG, FGF1, FGF2, FIGF, FLT1, JAG1, KDR,LAMA5, NRP1, NRP2, PGF, PLXDC1, STAB 1, VEGF, VEGFC, ANGPTL3, BAILCOL4A3, IL8, LAMA5, NRP1, NRP2, STAB 1, ANGPTL4, PECAM1, PF4, PROK2,SERPINF1, TNFAIP2, CCL11, CCL2, CXCL1, CXCL10, CXCL3, CXCL5, CXCL6,CXCL9, IFNA1, IFNB1, IFNG, IL1B, IL6, MDK, EDG1, EFNA1, EFNA3, EFNB2,EGF, EPHB4, FGFR3, HGF, IGF1, ITGB3, PDGFA, TEK, TGFA, TGFB1, TGFB2,TGFBR1, CCL2, CDH5, COL1A1, EDG1, ENG, ITGAV, ITGB3, THBS1, THBS2, BAD,BAG1, BCL2, CCNA1, CCNA2, CCND1, CCNE1, CCNE2, CDH1 (E-cadherin), CDKN1B(p27Kip1), CDKN2A (p161NK4a), COL6A1, CTNNB1 (b-catenin), CTSB(cathepsin B), ERBB2 (Her-2), ESR1, ESR2, F3 (TF), FOSL1 (FRA-1), GATA3,GSN (Gelsolin), IGFBP2, IL2RA, IL6, IL6R, IL6ST (glycoprotein 130),ITGA6 (a6 integrin), JUN, KLK5, KRT19, MAP2K7 (c-Jun), MKI67 (Ki-67),NGFB (GF), NGFR, NME1 (M23A), PGR, PLAU (uPA), PTEN, SERPINB5 (maspin),SERPINE1 (PAI-1), TGFA, THBS1 (thrombospondin-1), TIE (Tie-1), TNFRSF6(Fas), TNFSF6 (FasL), TOP2A (topoisomerase Iia), TP53, AZGP1(zinc-a-glycoprotein), BPAG1 (plectin), CDKN1A (p21Wap1/Cip1), CLDN7(claudin-7), CLU (clusterin), ERBB2 (Her-2), FGF1, FLRT1 (fibronectin),GABRP (GABAa), GNAS1, ID2, ITGA6 (a6 integrin), ITGB4 (b 4 integrin),KLFS (GC Box BP), KRT19 (Keratin 19), KRTHB6 (hair-specific type IIkeratin), MACMARCKS, MT3 (metallothionectin-III), MUC1 (mucin), PTGS2(COX-2), RAC2 (p21Rac2), S100A2, SCGB1D2 (lipophilin B), SCGB2A1(mammaglobin 2), SCGB2A2 (mammaglobin 1), SPRR1B (Spr1), THBS1, THBS2,THBS4, and TNFAIP2 (B94), RON, c-Met, CD64, DLL4, PLGF, CTLA4,phophatidylserine, ROBO4, CD80, CD22, CD40, CD23, CD28, CD80, CD55,CD38, CD70, CD74, CD30, CD138, CD56, CD33, CD2, CD137, DR4, DR5, RANKL,VEGFR2, PDGFR, VEGFR1, MTSP1, MSP, EPHB2, EPHA1, EPHA2, EpCAM, PGE2,NKG2D, LPA, SIP, APRIL, BCMA, MAPG, FLT3, PDGFR alpha, PDGFR beta, ROR1,PSMA, PSCA, SCD1, and CD59. To form the bispecific or trispecificantibodies of the invention, antibodies to any combination of theseantigens can be made; that is, each of these antigens can be optionallyand independently included or excluded from a multispecific antibodyaccording to the present invention.

Monoclonal antibody therapy has become an important therapeutic modalityfor treating autoimmune and inflammatory disorders (Chan & Carter, 2010,Nature Reviews Immunology 10:301-316; Reichert et al., 2005, NatureBiotechnology 23[9]:1073-1078; herein expressly incorporated byreference). Many proteins have been implicated in general autoimmune andinflammatory responses, and thus may be targeted by the immunogloublinsof the invention. Autoimmune and inflammatory targets include but arenot limited to C5, CCL1 (1-309), CCL11 (eotaxin), CCL13 (mcp-4), CCL15(MIP-1d), CCL16 (HCC-4), CCL17 (TARC), CCL18 (PARC), CCL19, CCL2(mcp-1), CCL20 (MIP-3a), CCL21 (MIP-2), CCL23 (MPIF-1), CCL24(MPIF-2/eotaxin-2), CCL25 (TECK), CCL26, CCL3 (MIP-1a), CCL4 (MIP-1b),CCL5 (RANTES), CCL7 (mcp-3), CCL8 (mcp-2), CXCL1, CXCL10 (IP-10), CXCL11(1-TAC/IP-9), CXCL12 (SDF1), CXCL13, CXCL14, CXCL2, CXCL3, CXCL5(ENA-78/LIX), CXCL6 (GCP-2), CXCL5, IL13, IL8, CCL13 (mcp-4), CCR1,CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CX3CR1, IL8RA, XCR1(CCXCR1), IFNA2, IL10, IL13, IL17C, ILIA, IL1B, IL1F10, IL1F5, IL1F6,IL1F7, IL1F8, IL1F9, IL22, IL5, IL8, IL9, LTA, LTB, MIF, SCYE1(endothelial Monocyte-activating cytokine), SPP1, TNF, TNFSF5, IFNA2,IL10RA, IL10RB, IL13, IL13RA1, IL5RA, IL9, IL9R, ABCF1, BCL6, C3, C4A,CEBPB, CRP, ICEBERG, IL1R1, IL1RN, IL8RB, LTB4R, TOLLIP, FADD, IRAK1,IRAK2, MYD88, NCK2, TNFAIP3, TRADD, TRAF1, TRAF2, TRAF3, TRAF4, TRAF5,TRAF6, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRL1, CD28, CD3E, CD3G, CD3Z,CD69, CD80, CD86, CNR1, CTLA4, CYSLTR1, FCER1A, FCER2, FCGR3A, GPR44,HAVCR2, OPRD1, P2RX7, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9,TLR10, BLR1, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL13,CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24,CCL25, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CX3CL1,CX3CR1, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL10, CXCL11, CXCL12,CXCL13, CXCR4, GPR2, SCYE1, SDF2, XCL1, XCL2, XCR1, AMH, AMHR2, BMPR1A,BMPR1B, BMPR2, C19orf10 (IL27w), CER1, CSF1, CSF2, CSF3, DKFZp451J0118,FGF2, GFI1, IFNA1, IFNB1, IFNG, IGF1, ILIA, IL1B, IL1R1, IL1R2, IL2,IL2RA, IL2RB, IL2RG, IL3, IL4, IL4R, IL5, IL5RA, IL6, IL6R, IL6ST, IL7,IL8, IL8RA, IL8RB, IL9, IL9R, IL10, IL10RA, IL10RB, IL11, IL12RA, IL12A,IL12B, IL12RB1, IL12RB2, IL13, IL13RA1, IL13RA2, IL15, IL15RA, IL16,IL17, IL17R, IL18, IL18R1, IL19, IL20, KITLG, LEP, LTA, LTB, LTB4R,LTB4R2, LTBR, MIF, NPPB, PDGFB, TBX21, TDGF1, TGFA, TGFB1, TGFB1I1,TGFB2, TGFB3, TGFB1, TGFBR1, TGFBR2, TGFBR3, TH1L, TNF, TNFRSF1A,TNFRSF1B, TNFRSF7, TNFRSF8, TNFRSF9, TNFRSF11A, TNFRSF21, TNFSF4,TNFSF5, TNFSF6, TNFSF11, VEGF, ZFPM2, and RNF110 (ZNF144). To form thebispecific or trispecific antibodies of the invention, antibodies to anycombination of these antigens can be made; that is, each of theseantigens can be optionally and independently included or excluded from amultispecific antibody according to the present invention.

Exemplary co-targets for autoimmune and inflammatory disorders includebut are not limited to IL-1 and TNFalpha, IL-6 and TNFalpha, IL-6 andIL-1, IgE and IL-13, IL-1 and IL-13, IL-4 and IL-13, IL-5 and IL-13,IL-9 and IL-13, CD19 and FcγRIIb, and CD79 and FcγRIIb.

Immunglobulins of the invention with specificity for the following pairsof targets to treat inflammatory disease are contemplated: TNF andIL-17A; TNF and RANKL; TNF and VEGF; TNF and SOST; TNF and DKK; TNF andalphaVbeta3; TNF and NGF; TNF and IL-23p19; TNF and IL-6; TNF and SOST;TNF and IL-6R; TNF and CD-20; IgE and IL-13; IL-13 and IL23p19; IgE andIL-4; IgE and IL-9; IgE and IL-9; IgE and IL-13; IL-13 and IL-9; IL-13and IL-4; IL-13 and IL-9; IL-13 and IL-9; IL-13 and IL-4; IL-13 andIL-23p19; IL-13 and IL-9; IL-6R and VEGF; IL-6R and IL-17A; IL-6R andRANKL; IL-17A and IL-1beta; IL-1beta and RANKL; IL-1beta and VEGF; RANKLand CD-20; IL-1alpha and IL-1beta; IL-1alpha and IL-1beta.

Pairs of targets that the immunoglobulins described herein can bind andbe useful to treat asthma may be determined. In an embodiment, suchtargets include, but are not limited to, IL-13 and IL-1beta, sinceIL-1beta is also implicated in inflammatory response in asthma; IL-13and cytokines and chemokines that are involved in inflammation, such asIL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-5; IL-13 and IL-25; IL-13and TARC; IL-13 and MDC; IL-13 and MIF; IL-13 and TGF-β; IL-13 and LHRagonist; IL-13 and CL25; IL-13 and SPRR2a; IL-13 and SPRR2b; and IL-13and ADAMS. The immunoglobulins herein may have specifity for one or moretargets involved in asthma selected from the group consisting of CSF1(MCSF), CSF2 (GM-CSF), CSF3 (GCSF), FGF2, IFNA1, IFNB1, IFNG, histamineand histamine receptors, ILIA, IL1B, IL2, IL3, IL4, IL5, IL6, IL7, IL8,IL9, IL10, IL11, IL12A, IL12B, IL13, IL14, IL15, IL16, IL17, IL18, IL19,KITLG, PDGFB, IL2RA, IL4R, IL5RA, IL8RA, IL8RB, IL12RB1, IL12RB2,IL13RA1, IL13RA2, IL18R1, TSLP, CCLi, CCL2, CCL3, CCL4, CCL5, CCL7,CCL8, CCL13, CCL17, CCL18, CCL19, CCL20, CCL22, CCL24, CX3CL1, CXCL1,CXCL2, CXCL3, XCLi, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CX3CR1,GPR2, XCR1, FOS, GATA3, JAK1, JAK3, STATE, TBX21, TGFB1, TNF, TNFSF6,YY1, CYSLTR1, FCER1A, FCER2, LTB4R, TB4R2, LTBR, and Chitinase. To formthe bispecific or trispecific antibodies of the invention, antibodies toany combination of these antigens can be made; that is, each of theseantigens can be optionally and independently included or excluded from amultispecific antibody according to the present invention.

Pairs of targets involved in rheumatoid arthritis (RA) may beco-targeted by the invention, including but not limited to TNF andIL-18; TNF and IL-12; TNF and IL-23; TNF and IL-1beta; TNF and MIF; TNFand IL-17; and TNF and IL-15.

Antigens that may be targeted in order to treat systemic lupuserythematosus (SLE) by the immunoglobulins herein include but are notlimited to CD-20, CD-22, CD-19, CD28, CD4, CD80, HLA-DRA, IL10, IL2,IL4, TNFRSF5, TNFRSF6, TNFSF5, TNFSF6, BLR1, HDAC4, HDAC5, HDAC7A,HDAC9, ICOSL, IGBP1, MS4A1, RGSI, SLA2, CD81, IFNB1, IL10, TNFRSF5,TNFRSF7, TNFSF5, AICDA, BLNK, GALNAC4S-6ST, HDAC4, HDAC5, HDAC7A, HDAC9,IL10, IL11, IL4, INHA, INHBA, KLF6, TNFRSF7, CD28, CD38, CD69, CD80,CD83, CD86, DPP4, FCER2, IL2RA, TNFRSF5, TNFSF7, CD24, CD37, CD40, CD72,CD74, CD79A, CD79B, CR2, ILIR2, ITGA2, ITGA3, MS4A1, ST6GALI, CDIC,CHSTIO, HLA-A, HLA-DRA, and NT5E; CTLA4, B7.1, B7.2, BlyS, BAFF, C5,IL-4, IL-6, IL-10, IFN-α, and TNF-α. To form the bispecific ortrispecific antibodies of the invention, antibodies to any combinationof these antigens can be made; that is, each of these antigens can beoptionally and independently included or excluded from a multispecificantibody according to the present invention.

The immunoglobulins herein may target antigens for the treatment ofmultiple sclerosis (MS), including but not limited to IL-12, TWEAK,IL-23, CXCL13, CD40, CD40L, IL-18, VEGF, VLA-4, TNF, CD45RB, CD200,IFNgamma, GM-CSF, FGF, C5, CD52, and CCR2. An embodiment includesco-engagement of anti-IL-12 and TWEAK for the treatment of MS.

One aspect of the invention pertains to immunoglobulins capable ofbinding one or more targets involved in sepsis, in an embodiment twotargets, selected from the group consisting TNF, IL-1, MIF, IL-6, IL-8,IL-18, IL-12, IL-23, FasL, LPS, Toll-like receptors, TLR-4, tissuefactor, MIP-2, ADORA2A, CASP1, CASP4, IL-10, IL-1B, NFκB1, PROC,TNFRSFIA, CSF3, CCR3, ILIRN, MIF, NFκB1, PTAFR, TLR2, TLR4, GPR44,HMOX1, midkine, IRAK1, NFκB2, SERPINAL SERPINEL and TREM1. To form thebispecific or trispecific antibodies of the invention, antibodies to anycombination of these antigens can be made; that is, each of theseantigens can be optionally and independently included or excluded from amultispecific antibody according to the present invention.

In some cases, immunoglobulins herein may be directed against antigensfor the treatment of infectious diseases.

Antibodies for Engineering

In some embodiments, the heterodimeric engineering and multispecificengineering described herein is done with portions of therapeuticantibodies. A number of antibodies that are approved for use, inclinical trials, or in development may benefit from the pI variants ofthe present invention. These antibodies are herein referred to as“clinical products and candidates”. Thus in a preferred embodiment, theheterodimerization variants may find use in a range of clinical productsand candidates. For example the heterodimerization variants of thepresent invention may find use in an antibody that has an anti-CD3variable region and then another antibody components, e.g. the variabledomains, the CDRs, etc., of clinical antibodies including, but notlimited to, rituximab (Rituxan®, IDEC/Genentech/Roche) (see for exampleU.S. Pat. No. 5,736,137), a chimeric anti-CD20 antibody approved totreat Non-Hodgkin's lymphoma; HuMax-CD20, an anti-CD20 currently beingdeveloped by Genmab, an anti-CD20 antibody described in U.S. Pat. No.5,500,362, AME-133 (Applied Molecular Evolution), hA20 (Immunomedics,Inc.), HumaLYM (Intracel), and PRO70769 (PCT/US2003/040426, entitled“Immunoglobulin Variants and Uses Thereof”). A number of antibodies thattarget members of the family of epidermal growth factor receptors,including EGFR (ErbB-1), Her2/neu (ErbB-2), Her3 (ErbB-3), Her4(ErbB-4), may benefit from pI engineered constant region(s) of theinvention. For example the pI engineered constant region(s) of theinvention may find use in an antibody that is substantially similar totrastuzumab (Herceptin®, Genentech) (see for example U.S. Pat. No.5,677,171), a humanized anti-Her2/neu antibody approved to treat breastcancer; pertuzumab (rhuMab-2C4, Omnitarg™) currently being developed byGenentech; an anti-Her2 antibody described in U.S. Pat. No. 4,753,894;cetuximab (Erbitux®, Imclone) (U.S. Pat. No. 4,943,533; PCT WO96/40210), a chimeric anti-EGFR antibody in clinical trials for avariety of cancers; ABX-EGF (U.S. Pat. No. 6,235,883), currently beingdeveloped by Abgenix-Immunex-Amgen; HuMax-EGFr (U.S. Ser. No.10/172,317), currently being developed by Genmab; 425, EMD55900,EMD62000, and EMD72000 (Merck KGaA) (U.S. Pat. No. 5,558,864; Murthy etal. 1987, Arch Biochem Biophys. 252(2):549-60; Rodeck et al., 1987, JCell Biochem. 35(4):315-20; Kettleborough et al., 1991, Protein Eng.4(7):773-83); ICR62 (Institute of Cancer Research) (PCT WO 95/20045;Modjtahedi et al., 1993, J. Cell Biophys. 1993, 22(1-3):129-46;Modjtahedi et al., 1993, Br J Cancer. 1993, 67(2):247-53; Modjtahedi etal, 1996, Br J Cancer, 73(2):228-35; Modjtahedi et al, 2003, Int JCancer, 105(2):273-80); TheraCIM hR3 (YM Biosciences, Canada and Centrode Immunologia Molecular, Cuba (U.S. Pat. No. 5,891,996; U.S. Pat. No.6,506,883; Mateo et al, 1997, Immunotechnology, 3(1):71-81); mAb-806(Ludwig Institue for Cancer Research, Memorial Sloan-Kettering)(Jungbluth et al. 2003, Proc Natl Acad Sci USA. 100(2):639-44); KSB-102(KS Biomedix); MR1-1 (IVAX, National Cancer Institute) (PCT WO0162931A2); and SC100 (Scancell) (PCT WO 01/88138). In another preferredembodiment, the pI engineered constant region(s) of the presentinvention may find use in alemtuzumab (Campath®, Millenium), a humanizedmonoclonal antibody currently approved for treatment of B-cell chroniclymphocytic leukemia. The pI engineered constant region(s) of thepresent invention may find use in a variety of antibodies that aresubstantially similar to other clinical products and candidates,including but not limited to muromonab-CD3 (Orthoclone OKT3®), ananti-CD3 antibody developed by Ortho Biotech/Johnson & Johnson,ibritumomab tiuxetan (Zevalin®), an anti-CD20 antibody developed byIDEC/Schering AG, gemtuzumab ozogamicin (Mylotarg®), an anti-CD33 (p67protein) antibody developed by Celltech/Wyeth, alefacept (Amevive®), ananti-LFA-3 Fc fusion developed by Biogen), abciximab (ReoPro®),developed by Centocor/Lilly, basiliximab (Simulect®), developed byNovartis, palivizumab (Synagis®), developed by MedImmune, infliximab(Remicade®), an anti-TNFalpha antibody developed by Centocor, adalimumab(Humira®), an anti-TNFalpha antibody developed by Abbott, Humicade™, ananti-TNFalpha antibody developed by Celltech, etanercept (Enbrel®), ananti-TNFalpha Fc fusion developed by Immunex/Amgen, ABX-CBL, ananti-CD147 antibody being developed by Abgenix, ABX-IL8, an anti-IL8antibody being developed by Abgenix, ABX-MA1, an anti-MUC18 antibodybeing developed by Abgenix, Pemtumomab (R1549, 90Y-muHMFG1), ananti-MUC1 In development by Antisoma, Therex (R1550), an anti-MUC1antibody being developed by Antisoma, AngioMab (AS1405), being developedby Antisoma, HuBC-1, being developed by Antisoma, Thioplatin (AS1407)being developed by Antisoma, Antegren® (natalizumab), ananti-alpha-4-beta-1 (VLA-4) and alpha-4-beta-7 antibody being developedby Biogen, VLA-1 mAb, an anti-VLA-1 integrin antibody being developed byBiogen, LTBR mAb, an anti-lymphotoxin beta receptor (LTBR) antibodybeing developed by Biogen, CAT-152, an anti-TGF-β2 antibody beingdeveloped by Cambridge Antibody Technology, J695, an anti-IL-12 antibodybeing developed by Cambridge Antibody Technology and Abbott, CAT-192, ananti-TGFβ1 antibody being developed by Cambridge Antibody Technology andGenzyme, CAT-213, an anti-Eotaxin1 antibody being developed by CambridgeAntibody Technology, LymphoStat-B™ an anti-Blys antibody being developedby Cambridge Antibody Technology and Human Genome Sciences Inc.,TRAIL-R1mAb, an anti-TRAIL-R1 antibody being developed by CambridgeAntibody Technology and Human Genome Sciences, Inc., Avastin™(bevacizumab, rhuMAb-VEGF), an anti-VEGF antibody being developed byGenentech, an anti-HER receptor family antibody being developed byGenentech, Anti-Tissue Factor (ATF), an anti-Tissue Factor antibodybeing developed by Genentech, Xolair™ (Omalizumab), an anti-IgE antibodybeing developed by Genentech, Raptiva™ (Efalizumab), an anti-CD11aantibody being developed by Genentech and Xoma, MLN-02 Antibody(formerly LDP-02), being developed by Genentech and MilleniumPharmaceuticals, HuMax CD4, an anti-CD4 antibody being developed byGenmab, HuMax-IL15, an anti-IL15 antibody being developed by Genmab andAmgen, HuMax-Inflam, being developed by Genmab and Medarex,HuMax-Cancer, an anti-Heparanase I antibody being developed by Genmaband Medarex and Oxford GcoSciences, HuMax-Lymphoma, being developed byGenmab and Amgen, HuMax-TAC, being developed by Genmab, IDEC-131, andanti-CD40L antibody being developed by IDEC Pharmaceuticals, IDEC-151(Clenoliximab), an anti-CD4 antibody being developed by IDECPharmaceuticals, IDEC-114, an anti-CD80 antibody being developed by IDECPharmaceuticals, IDEC-152, an anti-CD23 being developed by IDECPharmaceuticals, anti-macrophage migration factor (MIF) antibodies beingdeveloped by IDEC Pharmaceuticals, BEC2, an anti-idiotypic antibodybeing developed by Imclone, IMC-1C11, an anti-KDR antibody beingdeveloped by Imclone, DC101, an anti-flk-1 antibody being developed byImclone, anti-VE cadherin antibodies being developed by Imclone,CEA-Cide™ (labetuzumab), an anti-carcinoembryonic antigen (CEA) antibodybeing developed by Immunomedics, LymphoCide™ (Epratuzumab), an anti-CD22antibody being developed by Immunomedics, AFP-Cide, being developed byImmunomedics, MyelomaCide, being developed by Immunomedics, LkoCide,being developed by Immunomedics, ProstaCide, being developed byImmunomedics, MDX-010, an anti-CTLA4 antibody being developed byMedarex, MDX-060, an anti-CD30 antibody being developed by Medarex,MDX-070 being developed by Medarex, MDX-018 being developed by Medarex,Osidem™ (IDM-1), and anti-Her2 antibody being developed by Medarex andImmuno-Designed Molecules, HuMax™-CD4, an anti-CD4 antibody beingdeveloped by Medarex and Genmab, HuMax-IL15, an anti-IL15 antibody beingdeveloped by Medarex and Genmab, CNTO 148, an anti-TNFα antibody beingdeveloped by Medarex and Centocor/J&J, CNTO 1275, an anti-cytokineantibody being developed by Centocor/J&J, MOR101 and MOR102,anti-intercellular adhesion molecule-1 (ICAM-1) (CD54) antibodies beingdeveloped by MorphoSys, MOR201, an anti-fibroblast growth factorreceptor 3 (FGFR-3) antibody being developed by MorphoSys, Nuvion®(visilizumab), an anti-CD3 antibody being developed by Protein DesignLabs, HuZAF™, an anti-gamma interferon antibody being developed byProtein Design Labs, Anti-α5β1 Integrin, being developed by ProteinDesign Labs, anti-IL-12, being developed by Protein Design Labs, ING-1,an anti-Ep-CAM antibody being developed by Xoma, and MLN01, ananti-Beta2 integrin antibody being developed by Xoma, an pI-ADC antibodybeing developed by Seattle Genetics, all of the above-cited referencesin this paragraph are expressly incorporated herein by reference.

The antibodies of the present invention are generally isolated orrecombinant. “Isolated,” when used to describe the various polypeptidesdisclosed herein, means a polypeptide that has been identified andseparated and/or recovered from a cell or cell culture from which it wasexpressed. Ordinarily, an isolated polypeptide will be prepared by atleast one purification step. An “isolated antibody,” refers to anantibody which is substantially free of other antibodies havingdifferent antigenic specificities.

“Specific binding” or “specifically binds to” or is “specific for” aparticular antigen or an epitope means binding that is measurablydifferent from a non-specific interaction. Specific binding can bemeasured, for example, by determining binding of a molecule compared tobinding of a control molecule, which generally is a molecule of similarstructure that does not have binding activity. For example, specificbinding can be determined by competition with a control molecule that issimilar to the target.

Specific binding for a particular antigen or an epitope can beexhibited, for example, by an antibody having a KD for an antigen orepitope of at least about 10-4 M, at least about 10-5 M, at least about10-6 M, at least about 10-7 M, at least about 10-8 M, at least about10-9 M, alternatively at least about 10-10 M, at least about 10-11 M, atleast about 10-12 M, or greater, where KD refers to a dissociation rateof a particular antibody-antigen interaction. Typically, an antibodythat specifically binds an antigen will have a KD that is 20-, 50-,100-, 500-, 1000-, 5,000-, 10,000- or more times greater for a controlmolecule relative to the antigen or epitope.

Also, specific binding for a particular antigen or an epitope can beexhibited, for example, by an antibody having a KA or Ka for an antigenor epitope of at least 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- ormore times greater for the epitope relative to a control, where KA or Karefers to an association rate of a particular antibody-antigeninteraction.

Antibody-Drug Conjugates

In some embodiments, the multispecific antibodies of the invention areconjugated with drugs to form antibody-drug conjugates (ADCs). Ingeneral, ADCs are used in oncology applications, where the use ofantibody-drug conjugates for the local delivery of cytotoxic orcytostatic agents allows for the targeted delivery of the drug moiety totumors, which can allow higher efficacy, lower toxicity, etc. Anoverview of this technology is provided in Ducry et al., BioconjugateChem., 21:5-13 (2010), Carter et al., Cancer J. 14(3):154 (2008) andSenter, Current Opin. Chem. Biol. 13:235-244 (2009), all of which arehereby incorporated by reference in their entirety.

Thus the invention provides multispecific antibodies conjugated todrugs. Generally, conjugation is done by covalent attachment to theantibody, as further described below, and generally relies on a linker,often a peptide linkage (which, as described below, may be designed tobe sensitive to cleavage by proteases at the target site or not). Inaddition, as described above, linkage of the linker-drug unit (LU-D) canbe done by attachment to cysteines within the antibody. As will beappreciated by those in the art, the number of drug moieties perantibody can change, depending on the conditions of the reaction, andcan vary from 1:1 to 10:1 drug:antibody. As will be appreciated by thosein the art, the actual number is an average.

Thus the invention provides multispecific antibodies conjugated todrugs. As described below, the drug of the ADC can be any number ofagents, including but not limited to cytotoxic agents such aschemotherapeutic agents, growth inhibitory agents, toxins (for example,an enzymatically active toxin of bacterial, fungal, plant, or animalorigin, or fragments thereof), or a radioactive isotope (that is, aradioconjugate) are provided. In other embodiments, the inventionfurther provides methods of using the ADCs.

Drugs for use in the present invention include cytotoxic drugs,particularly those which are used for cancer therapy. Such drugsinclude, in general, DNA damaging agents, anti-metabolites, naturalproducts and their analogs. Exemplary classes of cytotoxic agentsinclude the enzyme inhibitors such as dihydrofolate reductaseinhibitors, and thymidylate synthase inhibitors, DNA intercalators, DNAcleavers, topoisomerase inhibitors, the anthracycline family of drugs,the vinca drugs, the mitomycins, the bleomycins, the cytotoxicnucleosides, the pteridine family of drugs, diynenes, thepodophyllotoxins, dolastatins, maytansinoids, differentiation inducers,and taxols.

Members of these classes include, for example, methotrexate,methopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine,cytosine arabinoside, melphalan, leurosine, leurosideine, actinomycin,daunorubicin, doxorubicin, mitomycin C, mitomycin A, caminomycin,aminopterin, tallysomycin, podophyllotoxin and podophyllotoxinderivatives such as etoposide or etoposide phosphate, vinblastine,vincristine, vindesine, taxanes including taxol, taxotere retinoic acid,butyric acid, N8-acetyl spermidine, camptothecin, calicheamicin,esperamicin, ene-diynes, duocarmycin A, duocarmycin SA, calicheamicin,camptothecin, maytansinoids (including DM1), monomethylauristatin E(MMAE), monomethylauristatin F (MMAF), and maytansinoids (DM4) and theiranalogues.

Toxins may be used as antibody-toxin conjugates and include bacterialtoxins such as diphtheria toxin, plant toxins such as ricin, smallmolecule toxins such as geldanamycin (Mandler et al (2000) J. Nat.Cancer Inst. 92(19):1573-1581; Mandler et al (2000) Bioorganic & Med.Chem. Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem.13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl.Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al (1998)Cancer Res. 58:2928; Hinman et al (1993) Cancer Res. 53:3336-3342).Toxins may exert their cytotoxic and cytostatic effects by mechanismsincluding tubulin binding, DNA binding, or topoisomerase inhibition.

Conjugates of a multispecific antibody and one or more small moleculetoxins, such as a maytansinoids, dolastatins, auristatins, atrichothecene, calicheamicin, and CC1065, and the derivatives of thesetoxins that have toxin activity, are contemplated.

Maytansinoids

Maytansine compounds suitable for use as maytansinoid drug moieties arewell known in the art, and can be isolated from natural sourcesaccording to known methods, produced using genetic engineeringtechniques (see Yu et al (2002) PNAS 99:7968-7973), or maytansinol andmaytansinol analogues prepared synthetically according to known methods.As described below, drugs may be modified by the incorporation of afunctionally active group such as a thiol or amine group for conjugationto the antibody.

Exemplary maytansinoid drug moieties include those having a modifiedaromatic ring, such as: C-19-dechloro (U.S. Pat. No. 4,256,746)(prepared by lithium aluminum hydride reduction of ansamytocin P2);C-20-hydroxy (or C-20-demethyl) +/−C-19-dechloro (U.S. Pat. Nos.4,361,650 and 4,307,016) (prepared by demethylation using Streptomycesor Actinomyces or dechlorination using LAH); and C-20-demethoxy,C-20-acyloxy (—OCOR), +/−dechloro (U.S. Pat. No. 4,294,757) (prepared byacylation using acyl chlorides) and those having modifications at otherpositions

Exemplary maytansinoid drug moieties also include those havingmodifications such as: C-9-SH (U.S. Pat. No. 4,424,219) (prepared by thereaction of maytansinol with H2S or P2S5);C-14-alkoxymethyl(demethoxy/CH2OR) (U.S. Pat. No. 4,331,598);C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH2OAc) (U.S. Pat. No.4,450,254) (prepared from Nocardia); C-15-hydroxy/acyloxy (U.S. Pat. No.4,364,866) (prepared by the conversion of maytansinol by Streptomyces);C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (isolated fromTrewia nudlflora); C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and4,322,348) (prepared by the demethylation of maytansinol byStreptomyces); and 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared by thetitanium trichloride/LAH reduction of maytansinol).

Of particular use are DM1 (disclosed in U.S. Pat. No. 5,208,020,incorporated by reference) and DM4 (disclosed in U.S. Pat. No.7,276,497, incorporated by reference). See also a number of additionalmaytansinoid derivatives and methods in U.S. Pat. No. 5,416,064,WO/01/24763, U.S. Pat. No. 7,303,749, U.S. Pat. No. 7,601,354, U.S. Ser.No. 12/631,508, WO02/098883, U.S. Pat. No. 6,441,163, U.S. Pat. No.7,368,565, WO02/16368 and WO04/1033272, all of which are expresslyincorporated by reference in their entirety.

ADCs containing maytansinoids, methods of making same, and theirtherapeutic use are disclosed, for example, in U.S. Pat. Nos. 5,208,020;5,416,064; 6,441,163 and European Patent EP 0 425 235 B1, thedisclosures of which are hereby expressly incorporated by reference. Liuet al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) described ADCscomprising a maytansinoid designated DM1 linked to the monoclonalantibody C242 directed against human colorectal cancer. The conjugatewas found to be highly cytotoxic towards cultured colon cancer cells,and showed antitumor activity in an in vivo tumor growth assay.

Chari et al., Cancer Research 52:127-131 (1992) describe ADCs in which amaytansinoid was conjugated via a disulfide linker to the murineantibody A7 binding to an antigen on human colon cancer cell lines, orto another murine monoclonal antibody TA.1 that binds the HER-2/neuoncogene. The cytotoxicity of the TA.1-maytansonoid conjugate was testedin vitro on the human breast cancer cell line SK-BR-3, which expresses3×105 HER-2 surface antigens per cell. The drug conjugate achieved adegree of cytotoxicity similar to the free maytansinoid drug, whichcould be increased by increasing the number of maytansinoid moleculesper antibody molecule. The A7-maytansinoid conjugate showed low systemiccytotoxicity in mice.

Auristatins and Dolastatins

In some embodiments, the ADC comprises a multispecific antibodyconjugated to dolastatins or dolostatin peptidic analogs andderivatives, the auristatins (U.S. Pat. Nos. 5,635,483; 5,780,588).Dolastatins and auristatins have been shown to interfere withmicrotubule dynamics, GTP hydrolysis, and nuclear and cellular division(Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584)and have anticancer (U.S. Pat. No. 5,663,149) and antifungal activity(Pettit et al (1998) Antimicrob. Agents Chemother. 42:2961-2965). Thedolastatin or auristatin drug moiety may be attached to the antibodythrough the N (amino) terminus or the C (carboxyl) terminus of thepeptidic drug moiety (WO 02/088172).

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties DE and DF, disclosed in “Senter etal, Proceedings of the American Association for Cancer Research, Volume45, Abstract Number 623, presented Mar. 28, 2004 and described in UnitedStates Patent Publication No. 2005/0238648, the disclosure of which isexpressly incorporated by reference in its entirety.

An exemplary auristatin embodiment is MMAE (see U.S. Pat. No. 6,884,869expressly incorporated by reference in its entirety).

Another exemplary auristatin embodiment is MMAF (see US 2005/0238649,U.S. Pat. No. 5,767,237 and U.S. Pat. No. 6,124,431, expresslyincorporated by reference in their entirety).

Additional exemplary embodiments comprising MMAE or MMAF and variouslinker components (described further herein) have the followingstructures and abbreviations (wherein Ab means antibody and p is 1 toabout 8):

Typically, peptide-based drug moieties can be prepared by forming apeptide bond between two or more amino acids and/or peptide fragments.Such peptide bonds can be prepared, for example, according to the liquidphase synthesis method (see E. Schroder and K. Lubke, “The Peptides”,volume 1, pp 76-136, 1965, Academic Press) that is well known in thefield of peptide chemistry. The auristatin/dolastatin drug moieties maybe prepared according to the methods of: U.S. Pat. No. 5,635,483; U.S.Pat. No. 5,780,588; Pettit et al (1989) J. Am. Chem. Soc. 111:5463-5465;Pettit et al (1998) Anti-Cancer Drug Design 13:243-277; Pettit, G. R.,et al. Synthesis, 1996, 719-725; Pettit et al (1996) J. Chem. Soc.Perkin Trans. 1 5:859-863; and Doronina (2003) Nat Biotechnol21(7):778-784.

Calicheamicin

In other embodiments, the ADC comprises an antibody of the inventionconjugated to one or more calicheamicin molecules. For example, Mylotargis the first commercial ADC drug and utilizes calicheamicin yl as thepayload (see U.S. Pat. No. 4,970,198, incorporated by reference in itsentirety). Additional calicheamicin derivatives are described in U.S.Pat. Nos. 5,264,586, 5,384,412, 5,550,246, 5,739,116, 5,773,001,5,767,285 and 5,877,296, all expressly incorporated by reference. Thecalicheamicin family of antibiotics are capable of producingdouble-stranded DNA breaks at sub-picomolar concentrations. For thepreparation of conjugates of the calicheamicin family, see U.S. Pat.Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710,5,773,001, 5,877,296 (all to American Cyanamid Company). Structuralanalogues of calicheamicin which may be used include, but are notlimited to, γ1I, α2I, α2I, N-acetyl-γ1I, PSAG and θI1 (Hinman et al.,Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research58:2925-2928 (1998) and the aforementioned U.S. patents to AmericanCyanamid). Another anti-tumor drug that the antibody can be conjugatedis QFA which is an antifolate. Both calicheamicin and QFA haveintracellular sites of action and do not readily cross the plasmamembrane. Therefore, cellular uptake of these agents through antibodymediated internalization greatly enhances their cytotoxic effects.

Duocarmycins

CC-1065 (see 4,169,888, incorporated by reference) and duocarmycins aremembers of a family of antitumor antibiotics utilized in ADCs. Theseantibiotics appear to work through sequence-selectively alkylating DNAat the N3 of adenine in the minor groove, which initiates a cascade ofevents that result in apoptosis.

Important members of the duocarmycins include duocarmycin A (U.S. Pat.No. 4,923,990, incorporated by reference) and duocarmycin SA (U.S. Pat.No. 5,101,038, incorporated by reference), and a large number ofanalogues as described in U.S. Pat. Nos. 7,517,903, 7,691,962,5,101,038; 5,641,780; 5,187,186; 5,070,092; 5,070,092; 5,641,780;5,101,038; 5,084,468, 5,475,092, 5,585,499, 5,846,545, WO2007/089149,WO2009/017394A1, 5,703,080, 6,989,452, 7,087,600, 7,129,261, 7,498,302,and 7,507,420, all of which are expressly incorporated by reference.

Other Cytotoxic Agents

Other antitumor agents that can be conjugated to the antibodies of theinvention include BCNU, streptozoicin, vincristine and 5-fluorouracil,the family of agents known collectively LL-E33288 complex described inU.S. Pat. Nos. 5,053,394, 5,770,710, as well as esperamicins (U.S. Pat.No. 5,877,296).

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

The present invention further contemplates an ADC formed between anantibody and a compound with nucleolytic activity (e.g., a ribonucleaseor a DNA endonuclease such as a deoxyribonuclease; DNase).

For selective destruction of the tumor, the antibody may comprise ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of radioconjugated antibodies. Examples includeAt211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 andradioactive isotopes of Lu.

The radio- or other labels may be incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as Tc99m or 1123, Re186, Re188 and In111 can be attached viaa cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al (1978) Biochem.Biophys. Res. Commun 80: 49-57 can be used to incorporate Iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

For compositions comprising a plurality of antibodies, the drug loadingis represented by p, the average number of drug molecules per Antibody.Drug loading may range from 1 to 20 drugs (D) per Antibody. The averagenumber of drugs per antibody in preparation of conjugation reactions maybe characterized by conventional means such as mass spectroscopy, ELISAassay, and HPLC. The quantitative distribution ofAntibody-Drug-Conjugates in terms of p may also be determined.

In some instances, separation, purification, and characterization ofhomogeneous Antibody-Drug-conjugates where p is a certain value fromAntibody-Drug-Conjugates with other drug loadings may be achieved bymeans such as reverse phase HPLC or electrophoresis. In exemplaryembodiments, p is 2, 3, 4, 5, 6, 7, or 8 or a fraction thereof.

The generation of Antibody-drug conjugate compounds can be accomplishedby any technique known to the skilled artisan. Briefly, theAntibody-drug conjugate compounds can include a multispecific antibodyas the Antibody unit, a drug, and optionally a linker that joins thedrug and the binding agent.

A number of different reactions are available for covalent attachment ofdrugs and/or linkers to binding agents. This is can be accomplished byreaction of the amino acid residues of the binding agent, for example,antibody molecule, including the amine groups of lysine, the freecarboxylic acid groups of glutamic and aspartic acid, the sulfhydrylgroups of cysteine and the various moieties of the aromatic amino acids.A commonly used non-specific methods of covalent attachment is thecarbodiimide reaction to link a carboxy (or amino) group of a compoundto amino (or carboxy) groups of the antibody. Additionally, bifunctionalagents such as dialdehydes or imidoesters have been used to link theamino group of a compound to amino groups of an antibody molecule.

Also available for attachment of drugs to binding agents is the Schiffbase reaction. This method involves the periodate oxidation of a drugthat contains glycol or hydroxy groups, thus forming an aldehyde whichis then reacted with the binding agent. Attachment occurs via formationof a Schiff base with amino groups of the binding agent. Isothiocyanatescan also be used as coupling agents for covalently attaching drugs tobinding agents. Other techniques are known to the skilled artisan andwithin the scope of the present invention.

In some embodiments, an intermediate, which is the precursor of thelinker, is reacted with the drug under appropriate conditions. In otherembodiments, reactive groups are used on the drug and/or theintermediate. The product of the reaction between the drug and theintermediate, or the derivatized drug, is subsequently reacted with anmultispecific antibody of the invention under appropriate conditions.

It will be understood that chemical modifications may also be made tothe desired compound in order to make reactions of that compound moreconvenient for purposes of preparing conjugates of the invention. Forexample a functional group e g amine, hydroxyl, or sulfhydryl, may beappended to the drug at a position which has minimal or an acceptableeffect on the activity or other properties of the drug

Linker Units

Typically, the antibody-drug conjugate compounds comprise a Linker unitbetween the drug unit and the antibody unit. In some embodiments, thelinker is cleavable under intracellular or extracellular conditions,such that cleavage of the linker releases the drug unit from theantibody in the appropriate environment. For example, solid tumors thatsecrete certain proteases may serve as the target of the cleavablelinker; in other embodiments, it is the intracellular proteases that areutilized. In yet other embodiments, the linker unit is not cleavable andthe drug is released, for example, by antibody degradation in lysosomes.

In some embodiments, the linker is cleavable by a cleaving agent that ispresent in the intracellular environment (for example, within a lysosomeor endosome or caveolea). The linker can be, for example, a peptidyllinker that is cleaved by an intracellular peptidase or protease enzyme,including, but not limited to, a lysosomal or endosomal protease. Insome embodiments, the peptidyl linker is at least two amino acids longor at least three amino acids long or more.

Cleaving agents can include, without limitation, cathepsins B and D andplasmin, all of which are known to hydrolyze dipeptide drug derivativesresulting in the release of active drug inside target cells (see, e.g.,Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). Peptidyllinkers that are cleavable by enzymes that are present inCD38-expressing cells. For example, a peptidyl linker that is cleavableby the thiol-dependent protease cathepsin-B, which is highly expressedin cancerous tissue, can be used (e.g., a Phe-Leu or a Gly-Phe-Leu-Glylinker (SEQ ID NO: X)). Other examples of such linkers are described,e.g., in U.S. Pat. No. 6,214,345, incorporated herein by reference inits entirety and for all purposes.

In some embodiments, the peptidyl linker cleavable by an intracellularprotease is a Val-Cit linker or a Phe-Lys linker (see, e.g., U.S. Pat.No. 6,214,345, which describes the synthesis of doxorubicin with theval-cit linker).

In other embodiments, the cleavable linker is pH-sensitive, that is,sensitive to hydrolysis at certain pH values. Typically, thepH-sensitive linker hydrolyzable under acidic conditions. For example,an acid-labile linker that is hydrolyzable in the lysosome (for example,a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide,orthoester, acetal, ketal, or the like) may be used. (See, e.g., U.S.Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999,Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem.264:14653-14661.) Such linkers are relatively stable under neutral pHconditions, such as those in the blood, but are unstable at below pH 5.5or 5.0, the approximate pH of the lysosome. In certain embodiments, thehydrolyzable linker is a thioether linker (such as, e.g., a thioetherattached to the therapeutic agent via an acylhydrazone bond (see, e.g.,U.S. Pat. No. 5,622,929).

In yet other embodiments, the linker is cleavable under reducingconditions (for example, a disulfide linker). A variety of disulfidelinkers are known in the art, including, for example, those that can beformed using SATA (N-succinimidyl-5-acetylthioacetate), SPDP(N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB(N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT(N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene)-,SPDB and SMPT. (See, e.g., Thorpe et al., 1987, Cancer Res.47:5924-5931; Wawrzynczak et al., In Immunoconjugates: AntibodyConjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed.,Oxford U. Press, 1987. See also U.S. Pat. No. 4,880,935.)

In other embodiments, the linker is a malonate linker (Johnson et al.,1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau etal., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a 3′-N-amide analog(Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12).

In yet other embodiments, the linker unit is not cleavable and the drugis released by antibody degradation. (See U.S. Publication No.2005/0238649 incorporated by reference herein in its entirety and forall purposes).

In many embodiments, the linker is self-immolative. As used herein, theterm “self-immolative Spacer” refers to a bifunctional chemical moietythat is capable of covalently linking together two spaced chemicalmoieties into a stable tripartite molecule. It will spontaneouslyseparate from the second chemical moiety if its bond to the first moietyis cleaved. See for example, WO 2007059404A2, WO06110476A2,WO05112919A2, WO2010/062171, WO09/017394, WO07/089149, WO 07/018431,WO04/043493 and WO02/083180, which are directed to drug-cleavablesubstrate conjugates where the drug and cleavable substrate areoptionally linked through a self-immolative linker and which are allexpressly incorporated by reference.

Often the linker is not substantially sensitive to the extracellularenvironment. As used herein, “not substantially sensitive to theextracellular environment,” in the context of a linker, means that nomore than about 20%, 15%, 10%, 5%, 3%, or no more than about 1% of thelinkers, in a sample of antibody-drug conjugate compound, are cleavedwhen the antibody-drug conjugate compound presents in an extracellularenvironment (for example, in plasma).

Whether a linker is not substantially sensitive to the extracellularenvironment can be determined, for example, by incubating with plasmathe antibody-drug conjugate compound for a predetermined time period(for example, 2, 4, 8, 16, or 24 hours) and then quantitating the amountof free drug present in the plasma.

In other, non-mutually exclusive embodiments, the linker promotescellular internalization. In certain embodiments, the linker promotescellular internalization when conjugated to the therapeutic agent (thatis, in the milieu of the linker-therapeutic agent moiety of theantibody-drug conjugate compound as described herein). In yet otherembodiments, the linker promotes cellular internalization whenconjugated to both the auristatin compound and the multispecificantibodies of the invention.

A variety of exemplary linkers that can be used with the presentcompositions and methods are described in WO 2004-010957, U.S.Publication No. 2006/0074008, U.S. Publication No. 20050238649, and U.S.Publication No. 2006/0024317 (each of which is incorporated by referenceherein in its entirety and for all purposes).

Drug Loading

Drug loading is represented by p and is the average number of Drugmoieties per antibody in a molecule. Drug loading (“p”) may be 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or moremoieties (D) per antibody, although frequently the average number is afraction or a decimal. Generally, drug loading of from 1 to 4 isfrequently useful, and from 1 to 2 is also useful. ADCs of the inventioninclude collections of antibodies conjugated with a range of drugmoieties, from 1 to 20. The average number of drug moieties per antibodyin preparations of ADC from conjugation reactions may be characterizedby conventional means such as mass spectroscopy and, ELISA assay.

The quantitative distribution of ADC in terms of p may also bedetermined. In some instances, separation, purification, andcharacterization of homogeneous ADC where p is a certain value from ADCwith other drug loadings may be achieved by means such aselectrophoresis.

For some antibody-drug conjugates, p may be limited by the number ofattachment sites on the antibody. For example, where the attachment is acysteine thiol, as in the exemplary embodiments above, an antibody mayhave only one or several cysteine thiol groups, or may have only one orseveral sufficiently reactive thiol groups through which a linker may beattached. In certain embodiments, higher drug loading, e.g. p>5, maycause aggregation, insolubility, toxicity, or loss of cellularpermeability of certain antibody-drug conjugates. In certainembodiments, the drug loading for an ADC of the invention ranges from 1to about 8; from about 2 to about 6; from about 3 to about 5; from about3 to about 4; from about 3.1 to about 3.9; from about 3.2 to about 3.8;from about 3.2 to about 3.7; from about 3.2 to about 3.6; from about 3.3to about 3.8; or from about 3.3 to about 3.7. Indeed, it has been shownthat for certain ADCs, the optimal ratio of drug moieties per antibodymay be less than 8, and may be about 2 to about 5. See US 2005-0238649A1 (herein incorporated by reference in its entirety).

In certain embodiments, fewer than the theoretical maximum of drugmoieties are conjugated to an antibody during a conjugation reaction. Anantibody may contain, for example, lysine residues that do not reactwith the drug-linker intermediate or linker reagent, as discussed below.Generally, antibodies do not contain many free and reactive cysteinethiol groups which may be linked to a drug moiety; indeed most cysteinethiol residues in antibodies exist as disulfide bridges. In certainembodiments, an antibody may be reduced with a reducing agent such asdithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under partialor total reducing conditions, to generate reactive cysteine thiolgroups. In certain embodiments, an antibody is subjected to denaturingconditions to reveal reactive nucleophilic groups such as lysine orcysteine.

The loading (drug/antibody ratio) of an ADC may be controlled indifferent ways, e.g., by: (i) limiting the molar excess of drug-linkerintermediate or linker reagent relative to antibody, (ii) limiting theconjugation reaction time or temperature, (iii) partial or limitingreductive conditions for cysteine thiol modification, (iv) engineeringby recombinant techniques the amino acid sequence of the antibody suchthat the number and position of cysteine residues is modified forcontrol of the number and/or position of linker-drug attachements (suchas thioMab or thioFab prepared as disclosed herein and in WO2006/034488(herein incorporated by reference in its entirety)).

It is to be understood that where more than one nucleophilic groupreacts with a drug-linker intermediate or linker reagent followed bydrug moiety reagent, then the resulting product is a mixture of ADCcompounds with a distribution of one or more drug moieties attached toan antibody. The average number of drugs per antibody may be calculatedfrom the mixture by a dual ELISA antibody assay, which is specific forantibody and specific for the drug. Individual ADC molecules may beidentified in the mixture by mass spectroscopy and separated by HPLC,e.g. hydrophobic interaction chromatography.

In some embodiments, a homogeneous ADC with a single loading value maybe isolated from the conjugation mixture by electrophoresis orchromatography.

Methods of Determining Cytotoxic Effect of ADCs

Methods of determining whether a Drug or Antibody-Drug conjugate exertsa cytostatic and/or cytotoxic effect on a cell are known. Generally, thecytotoxic or cytostatic activity of an Antibody Drug conjugate can bemeasured by: exposing mammalian cells expressing a target protein of theAntibody Drug conjugate in a cell culture medium; culturing the cellsfor a period from about 6 hours to about 5 days; and measuring cellviability. Cell-based in vitro assays can be used to measure viability(proliferation), cytotoxicity, and induction of apoptosis (caspaseactivation) of the Antibody Drug conjugate.

For determining whether an Antibody Drug conjugate exerts a cytostaticeffect, a thymidine incorporation assay may be used. For example, cancercells expressing a target antigen at a density of 5,000 cells/well of a96-well plated can be cultured for a 72-hour period and exposed to 0.5μCi of 3H-thymidine during the final 8 hours of the 72-hour period. Theincorporation of 3H-thymidine into cells of the culture is measured inthe presence and absence of the Antibody Drug conjugate.

For determining cytotoxicity, necrosis or apoptosis (programmed celldeath) can be measured. Necrosis is typically accompanied by increasedpermeability of the plasma membrane; swelling of the cell, and ruptureof the plasma membrane. Apoptosis is typically characterized by membraneblebbing, condensation of cytoplasm, and the activation of endogenousendonucleases. Determination of any of these effects on cancer cellsindicates that an Antibody Drug conjugate is useful in the treatment ofcancers.

Cell viability can be measured by determining in a cell the uptake of adye such as neutral red, trypan blue, or ALAMAR™ blue (see, e.g., Pageet al., 1993, Intl. J. Oncology 3:473-476). In such an assay, the cellsare incubated in media containing the dye, the cells are washed, and theremaining dye, reflecting cellular uptake of the dye, is measuredspectrophotometrically. The protein-binding dye sulforhodamine B (SRB)can also be used to measure cytoxicity (Skehan et al., 1990, J. Natl.Cancer Inst. 82:1107-12).

Alternatively, a tetrazolium salt, such as MTT, is used in aquantitative colorimetric assay for mammalian cell survival andproliferation by detecting living, but not dead, cells (see, e.g.,Mosmann, 1983, J. Immunol. Methods 65:55-63).

Apoptosis can be quantitated by measuring, for example, DNAfragmentation. Commercial photometric methods for the quantitative invitro determination of DNA fragmentation are available. Examples of suchassays, including TUNEL (which detects incorporation of labelednucleotides in fragmented DNA) and ELISA-based assays, are described inBiochemica, 1999, no. 2, pp. 34-37 (Roche Molecular Biochemicals).

Apoptosis can also be determined by measuring morphological changes in acell. For example, as with necrosis, loss of plasma membrane integritycan be determined by measuring uptake of certain dyes (e.g., afluorescent dye such as, for example, acridine orange or ethidiumbromide). A method for measuring apoptotic cell number has beendescribed by Duke and Cohen, Current Protocols in Immunology (Coligan etal. eds., 1992, pp. 3.17.1-3.17.16). Cells also can be labeled with aDNA dye (e.g., acridine orange, ethidium bromide, or propidium iodide)and the cells observed for chromatin condensation and margination alongthe inner nuclear membrane. Other morphological changes that can bemeasured to determine apoptosis include, e.g., cytoplasmic condensation,increased membrane blebbing, and cellular shrinkage.

The presence of apoptotic cells can be measured in both the attached and“floating” compartments of the cultures. For example, both compartmentscan be collected by removing the supernatant, trypsinizing the attachedcells, combining the preparations following a centrifugation wash step(e.g., 10 minutes at 2000 rpm), and detecting apoptosis (e.g., bymeasuring DNA fragmentation). (See, e.g., Piazza et al., 1995, CancerResearch 55:3110-16).

In vivo, the effect of a therapeutic composition of the multispecificantibody of the invention can be evaluated in a suitable animal model.For example, xenogenic cancer models can be used, wherein cancerexplants or passaged xenograft tissues are introduced into immunecompromised animals, such as nude or SCID mice (Klein et al., 1997,Nature Medicine 3: 402-408). Efficacy can be measured using assays thatmeasure inhibition of tumor formation, tumor regression or metastasis,and the like.

Therapeutic Uses of Heterodimers

The multispecific proteins, particularly the multispecific antibodies ofthe present invention find use in a variety of therapeutic uses. Asdiscussed in FIG. 1 of Kontermann, supra, incorporated herein byreference, there are a number of dual targeting strategies for cancer,inflammation, etc.

Pharmaceutical Formulations, Administration and Dosing

The therapeutic compositions used in the practice of the foregoingmethods can be formulated into pharmaceutical compositions comprising acarrier suitable for the desired delivery method. Suitable carriersinclude any material that when combined with the therapeutic compositionretains the anti-tumor function of the therapeutic composition and isgenerally non-reactive with the patient's immune system. Examplesinclude, but are not limited to, any of a number of standardpharmaceutical carriers such as sterile phosphate buffered salinesolutions, bacteriostatic water, and the like (see, generally,Remington's Pharmaceutical Sciences 16th Edition, A. Osal., Ed., 1980).

Antibody Compositions for In Vivo Administration

Formulations of the antibodies used in accordance with the presentinvention are prepared for storage by mixing an antibody having thedesired degree of purity with optional pharmaceutically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. [1980]), in the form of lyophilizedformulations or aqueous solutions. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, it may be desirable to provide antibodies with otherspecifcities. Alternatively, or in addition, the composition maycomprise a cytotoxic agent, cytokine, growth inhibitory agent and/orsmall molecule antagonist. Such molecules are suitably present incombination in amounts that are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration should besterile, or nearly so. This is readily accomplished by filtrationthrough sterile filtration membranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and .gamma.ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods.

When encapsulated antibodies remain in the body for a long time, theymay denature or aggregate as a result of exposure to moisture at 37° C.,resulting in a loss of biological activity and possible changes inimmunogenicity. Rational strategies can be devised for stabilizationdepending on the mechanism involved. For example, if the aggregationmechanism is discovered to be intermolecular S—S bond formation throughthio-disulfide interchange, stabilization may be achieved by modifyingsulfhydryl residues, lyophilizing from acidic solutions, controllingmoisture content, using appropriate additives, and developing specificpolymer matrix compositions.

Administrative Modalities

The antibodies and chemotherapeutic agents of the invention areadministered to a subject, in accord with known methods, such asintravenous administration as a bolus or by continuous infusion over aperiod of time, by intramuscular, intraperitoneal, intracerobrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes. Intravenous or subcutaneousadministration of the antibody is preferred.

Treatment Modalities

In the methods of the invention, therapy is used to provide a positivetherapeutic response with respect to a disease or condition. By“positive therapeutic response” is intended an improvement in thedisease or condition, and/or an improvement in the symptoms associatedwith the disease or condition. For example, a positive therapeuticresponse would refer to one or more of the following improvements in thedisease: (1) a reduction in the number of neoplastic cells; (2) anincrease in neoplastic cell death; (3) inhibition of neoplastic cellsurvival; (5) inhibition (i.e., slowing to some extent, preferablyhalting) of tumor growth; (6) an increased patient survival rate; and(7) some relief from one or more symptoms associated with the disease orcondition.

Positive therapeutic responses in any given disease or condition can bedetermined by standardized response criteria specific to that disease orcondition. Tumor response can be assessed for changes in tumormorphology (i.e., overall tumor burden, tumor size, and the like) usingscreening techniques such as magnetic resonance imaging (MRI) scan,x-radiographic imaging, computed tomographic (CT) scan, bone scanimaging, endoscopy, and tumor biopsy sampling including bone marrowaspiration (BMA) and counting of tumor cells in the circulation.

In addition to these positive therapeutic responses, the subjectundergoing therapy may experience the beneficial effect of animprovement in the symptoms associated with the disease.

Thus for B cell tumors, the subject may experience a decrease in theso-called B symptoms, i.e., night sweats, fever, weight loss, and/orurticaria. For pre-malignant conditions, therapy with an multispecifictherapeutic agent may block and/or prolong the time before developmentof a related malignant condition, for example, development of multiplemyeloma in subjects suffering from monoclonal gammopathy of undeterminedsignificance (MGUS).

An improvement in the disease may be characterized as a completeresponse. By “complete response” is intended an absence of clinicallydetectable disease with normalization of any previously abnormalradiographic studies, bone marrow, and cerebrospinal fluid (CSF) orabnormal monoclonal protein in the case of myeloma.

Such a response may persist for at least 4 to 8 weeks, or sometimes 6 to8 weeks, following treatment according to the methods of the invention.Alternatively, an improvement in the disease may be categorized as beinga partial response. By “partial response” is intended at least about a50% decrease in all measurable tumor burden (i.e., the number ofmalignant cells present in the subject, or the measured bulk of tumormasses or the quantity of abnormal monoclonal protein) in the absence ofnew lesions, which may persist for 4 to 8 weeks, or 6 to 8 weeks.

Treatment according to the present invention includes a “therapeuticallyeffective amount” of the medicaments used. A “therapeutically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve a desired therapeutic result.

A therapeutically effective amount may vary according to factors such asthe disease state, age, sex, and weight of the individual, and theability of the medicaments to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the antibody or antibody portion areoutweighed by the therapeutically beneficial effects.

A “therapeutically effective amount” for tumor therapy may also bemeasured by its ability to stabilize the progression of disease. Theability of a compound to inhibit cancer may be evaluated in an animalmodel system predictive of efficacy in human tumors.

Alternatively, this property of a composition may be evaluated byexamining the ability of the compound to inhibit cell growth or toinduce apoptosis by in vitro assays known to the skilled practitioner. Atherapeutically effective amount of a therapeutic compound may decreasetumor size, or otherwise ameliorate symptoms in a subject. One ofordinary skill in the art would be able to determine such amounts basedon such factors as the subject's size, the severity of the subject'ssymptoms, and the particular composition or route of administrationselected.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. Parenteral compositions may beformulated in dosage unit form for ease of administration and uniformityof dosage. Dosage unit form as used herein refers to physically discreteunits suited as unitary dosages for the subjects to be treated; eachunit contains a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

The specification for the dosage unit forms of the present invention aredictated by and directly dependent on (a) the unique characteristics ofthe active compound and the particular therapeutic effect to beachieved, and (b) the limitations inherent in the art of compoundingsuch an active compound for the treatment of sensitivity in individuals.

The efficient dosages and the dosage regimens for the multispecificantibodies used in the present invention depend on the disease orcondition to be treated and may be determined by the persons skilled inthe art.

An exemplary, non-limiting range for a therapeutically effective amountof an multispecific antibody used in the present invention is about0.1-100 mg/kg, such as about 0.1-50 mg/kg, for example about 0.1-20mg/kg, such as about 0.1-10 mg/kg, for instance about 0.5, about such as0.3, about 1, or about 3 mg/kg. In another embodiment, the antibody isadministered in a dose of 1 mg/kg or more, such as a dose of from 1 to20 mg/kg, e.g. a dose of from 5 to 20 mg/kg, e.g. a dose of 8 mg/kg.

A medical professional having ordinary skill in the art may readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, a physician or a veterinarian couldstart doses of the medicament employed in the pharmaceutical compositionat levels lower than that required in order to achieve the desiredtherapeutic effect and gradually increase the dosage until the desiredeffect is achieved.

In one embodiment, the multispecific antibody is administered byinfusion in a weekly dosage of from 10 to 500 mg/kg such as of from 200to 400 mg/kg Such administration may be repeated, e.g., 1 to 8 times,such as 3 to 5 times. The administration may be performed by continuousinfusion over a period of from 2 to 24 hours, such as of from 2 to 12hours.

In one embodiment, the multispecific antibody is administered by slowcontinuous infusion over a long period, such as more than 24 hours, ifrequired to reduce side effects including toxicity.

In one embodiment the multispecific antibody is administered in a weeklydosage of from 250 mg to 2000 mg, such as for example 300 mg, 500 mg,700 mg, 1000 mg, 1500 mg or 2000 mg, for up to 8 times, such as from 4to 6 times. The administration may be performed by continuous infusionover a period of from 2 to 24 hours, such as of from 2 to 12 hours. Suchregimen may be repeated one or more times as necessary, for example,after 6 months or 12 months. The dosage may be determined or adjusted bymeasuring the amount of compound of the present invention in the bloodupon administration by for instance taking out a biological sample andusing anti-idiotypic antibodies which target the antigen binding regionof the multispecific antibody.

In a further embodiment, the multispecific antibody is administered onceweekly for 2 to 12 weeks, such as for 3 to 10 weeks, such as for 4 to 8weeks.

In one embodiment, the multispecific antibody is administered bymaintenance therapy, such as, e.g., once a week for a period of 6 monthsor more.

In one embodiment, the multispecific antibody is administered by aregimen including one infusion of an multispecific antibody followed byan infusion of an multispecific antibody conjugated to a radioisotope.The regimen may be repeated, e.g., 7 to 9 days later.

As non-limiting examples, treatment according to the present inventionmay be provided as a daily dosage of an antibody in an amount of about0.1-100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on atleast one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, or 40, or alternatively, at least one of week 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 afterinitiation of treatment, or any combination thereof, using single ordivided doses of every 24, 12, 8, 6, 4, or 2 hours, or any combinationthereof.

In some embodiments the multispecific antibody molecule thereof is usedin combination with one or more additional therapeutic agents, e.g. achemotherapeutic agent. Non-limiting examples of DNA damagingchemotherapeutic agents include topoisomerase I inhibitors (e.g.,irinotecan, topotecan, camptothecin and analogs or metabolites thereof,and doxorubicin); topoisomerase II inhibitors (e.g., etoposide,teniposide, and daunorubicin); alkylating agents (e.g., melphalan,chlorambucil, busulfan, thiotepa, ifosfamide, carmustine, lomustine,semustine, streptozocin, decarbazine, methotrexate, mitomycin C, andcyclophosphamide); DNA intercalators (e.g., cisplatin, oxaliplatin, andcarboplatin); DNA intercalators and free radical generators such asbleomycin; and nucleoside mimetics (e.g., 5-fluorouracil, capecitibine,gemcitabine, fludarabine, cytarabine, mercaptopurine, thioguanine,pentostatin, and hydroxyurea).

Chemotherapeutic agents that disrupt cell replication include:paclitaxel, docetaxel, and related analogs; vincristine, vinblastin, andrelated analogs; thalidomide, lenalidomide, and related analogs (e.g.,CC-5013 and CC-4047); protein tyrosine kinase inhibitors (e.g., imatinibmesylate and gefitinib); proteasome inhibitors (e.g., bortezomib); NF-κBinhibitors, including inhibitors of IκB kinase; antibodies which bind toproteins overexpressed in cancers and thereby downregulate cellreplication (e.g., trastuzumab, rituximab, cetuximab, and bevacizumab);and other inhibitors of proteins or enzymes known to be upregulated,over-expressed or activated in cancers, the inhibition of whichdownregulates cell replication.

In some embodiments, the antibodies of the invention can be used priorto, concurrent with, or after treatment with Velcade® (bortezomib).

All cited references are herein expressly incorporated by reference intheir entirety.

Whereas particular embodiments of the invention have been describedabove for purposes of illustration, it will be appreciated by thoseskilled in the art that numerous variations of the details may be madewithout departing from the invention as described in the appendedclaims.

EXAMPLES scFv Production

DNA encoding the anti-CD3 scFv with a C-terminal GlySer linker andHis₆-tag was generated by gene synthesis (Blue Heron Biotechnology,Bothell, Wash.) and was subcloned using standard molecular biologytechniques into the expression vector pTTS. Substitutions wereintroduced using either site-directed mutagenesis (QuikChange,Stratagene, Cedar Creek, Tex.) or additional gene synthesis andsubcloning using a BspEI restriction site placed in the (Gly₄Ser)₃ VH-VLlinker. DNA was transfected into HEK293E cells for expression andresulting proteins were purified from the supernatant using nickelaffinity (Qiagen, Valencia, Calif.) chromatography.

Determination of Tm

Differential scanning fluorimetry (DSF) experiments were performed usinga Bio-Rad CFX Connect Real-Time PCR Detection System. Proteins weremixed with SYPRO Orange fluorescent dye and diluted to 0.2 mg/mL in PBS.The final concentration of SYPRO Orange was 10×. After an initial 10minute incubation period at 25° C., proteins were heated from 25 to 95°C. using a heating rate of 1° C./min. A fluorescence measurement wastaken every 30 sec. Melting temperatures were calculated using theinstrument software. The results are shown in FIGS. 3A-3B.

Binding Studies: Human

Competition binding protocol: Binding of scFvs to CD3 was measured on Tcells in a competition experiment with labeled anti-CD3 antibody.Proteins at various concentrations were incubated on ice for 1 h with400 k PBMCs and PE-labeled anti-CD3 antibody (final concentration of 2μg/ml). FITC-labeled anti-CD4 (BD Biosciences, San Diego, Calif.) andAPC-labeled anti-CD8 (BioLegend, San Diego, Calif.) antibodies wereadded and incubated for an additional 30 min in 3% FBS/PBS. Samples werewashed twice, fixed with 1% paraformaldehyde/PBS and analyzed using aFACSCanto II flow cytometer (BD Biosciences). T cells were identified asCD4⁺ or CD8⁺ live lymphocytes; approximately 4,000-10,000 events werecaptured per sample. Binding of PE-labeled anti-CD3 antibody to T cellswas quantified by mean fluorescence intensity (MFI) values of CD4⁺ livelymphocytes, which were then plotted against the log of the scFvconcentrations. Data was fitted with a sigmoidal dose-response curveusing Prism 6 (GraphPad Software) and the relative affinities weredetermined as IC₅₀ values of each curve. Percentage fold improvementswere calculated from the H1_L1.4 scFv. Results are shown in FIGS. 4A-4B.

Binding Studies: Monkey

Binding of Fabs to CD3 was measured on cynomolgus monkey (specificallyMacaca fascicularis) T cells in a competition experiment with labeledanti-CD3 antibody. Proteins at various concentrations were incubated onice for 1.5 h with 200 k PBMCs and FITC-labeled anti-CD3 antibody (finalconcentration of 50 ng/mL). APC-labeled anti-CD4 (BD Biosciences, SanDiego, Calif.) antibody was added and incubated for an additional 1 hrin 3% FBS/PBS. Samples were washed twice, fixed with 1%paraformaldehyde/PBS and analyzed using a FACSCanto II flow cytometer(BD Biosciences). T cells were identified as CD4⁺ live lymphocytes;approximately 2,000-3,000 events were captured per sample. Binding ofFITC-labeled anti-CD3 antibody to T cells was quantified by meanfluorescence intensity (MFI) values of CD4⁺ live lymphocytes, which werethen plotted against the log of the Fab concentrations. Data was fittedwith a sigmoidal dose-response curve using Prism 6 (GraphPad Software)and the relative affinities were determined as IC₅₀ values of eachcurve.

1-33. (canceled)
 34. An anti-CD3 binding domain comprising a variableheavy sequence and a variable light sequence pair selected from thegroup consisting of SEQ ID NO:5 and SEQ ID NO:6; SEQ ID NO:9 and SEQ IDNO:10; SEQ ID NO:13 and SEQ ID NO:14; SEQ ID NO:17 and SEQ ID NO:18; SEQID NO:21 and SEQ ID NO:22; SEQ ID NO:25 and SEQ ID NO:26; SEQ ID NO:29and SEQ ID NO:30; SEQ ID NO:33 and SEQ ID NO:34; SEQ ID NO:37 and SEQ IDNO:38; SEQ ID NO:41 and SEQ ID NO:42; SEQ ID NO:45 and SEQ ID NO:46; SEQID NO:49 and SEQ ID NO:50; SEQ ID NO:53 and SEQ ID NO:54; SEQ ID NO:57and SEQ ID NO:58; SEQ ID NO:61 and SEQ ID NO:62; SEQ ID NO:65 and SEQ IDNO:66; SEQ ID NO:69 and SEQ ID NO:70; SEQ ID NO:73 and SEQ ID NO:74; SEQID NO:77 and SEQ ID NO:78; SEQ ID NO:81 and SEQ ID NO:82; SEQ ID NO:85and SEQ ID NO:86; SEQ ID NO:89 and SEQ ID NO:90; SEQ ID NO:93 and SEQ IDNO:94; SEQ ID NO:97 and SEQ ID NO:98; SEQ ID NO:101 and SEQ ID NO:102;SEQ ID NO:105 and SEQ ID NO:106; SEQ ID NO:109 and SEQ ID NO:110; SEQ IDNO:113 and SEQ ID NO:114; SEQ ID NO:117 and SEQ ID NO:118; SEQ ID NO:121and SEQ ID NO:122; SEQ ID NO:125 and SEQ ID NO:126; SEQ ID NO:129 andSEQ ID NO:130; SEQ ID NO:133 and SEQ ID NO:134; SEQ ID NO:137 and SEQ IDNO:138; SEQ ID NO:141 and SEQ ID NO:142; SEQ ID NO:145 and SEQ IDNO:146; SEQ ID NO:149 and SEQ ID NO:150; SEQ ID NO:153 and SEQ IDNO:154; SEQ ID NO:157 and SEQ ID NO:158; SEQ ID NO:161 and SEQ IDNO:162; SEQ ID NO:165 and SEQ ID NO:166; SEQ ID NO:169 and SEQ IDNO:170; SEQ ID NO:173 and SEQ ID NO:174; SEQ ID NO:177 and SEQ IDNO:178; SEQ ID NO:181 and SEQ ID NO:182; SEQ ID NO:185 and SEQ IDNO:186; SEQ ID NO:189 and SEQ ID NO:190; SEQ ID NO:193 and SEQ IDNO:194; SEQ ID NO:197 and SEQ ID NO:198; SEQ ID NO:201 and SEQ IDNO:202; SEQ ID NO:205 and SEQ ID NO:206; SEQ ID NO:209 and SEQ IDNO:210; SEQ ID NO:213 and SEQ ID NO:214; SEQ ID NO:217 and SEQ IDNO:218; SEQ ID NO:221 and SEQ ID NO:222; SEQ ID NO:225 and SEQ IDNO:226; SEQ ID NO:229 and SEQ ID NO:230; SEQ ID NO:233 and SEQ IDNO:234; SEQ ID NO:237 and SEQ ID NO:238; SEQ ID NO:241 and SEQ IDNO:242; SEQ ID NO:245 and SEQ ID NO:246; SEQ ID NO:249 and SEQ IDNO:250; SEQ ID NO:253 and SEQ ID NO:254; SEQ ID NO:257 and SEQ IDNO:258; SEQ ID NO:261 and SEQ ID NO:262; SEQ ID NO:265 and SEQ IDNO:266; SEQ ID NO:269 and SEQ ID NO:270; SEQ ID NO:273 and SEQ IDNO:274; SEQ ID NO:277 and SEQ ID NO:278; SEQ ID NO:281 and SEQ IDNO:282; SEQ ID NO:285 and SEQ ID NO:286; SEQ ID NO:289 and SEQ IDNO:290; SEQ ID NO:293 and SEQ ID NO:294; SEQ ID NO:297 and SEQ IDNO:298; SEQ ID NO:301 and SEQ ID NO:302; SEQ ID NO:305 and SEQ IDNO:306; SEQ ID NO:309 and SEQ ID NO:310; SEQ ID NO:313 and SEQ IDNO:314; SEQ ID NO:317 and SEQ ID NO:318; SEQ ID NO:321 and SEQ IDNO:322; SEQ ID NO:325 and SEQ ID NO:326; SEQ ID NO:329 and SEQ IDNO:330; SEQ ID NO:333 and SEQ ID NO:334; SEQ ID NO:337 and SEQ IDNO:338; SEQ ID NO:341 and SEQ ID NO:342; SEQ ID NO:345 and SEQ IDNO:346; SEQ ID NO:349 and SEQ ID NO:350; SEQ ID NO:353 and SEQ IDNO:354; SEQ ID NO:357 and SEQ ID NO:358; SEQ ID NO:361 and SEQ IDNO:362; SEQ ID NO:365 and SEQ ID NO:366; SEQ ID NO:369 and SEQ IDNO:370; SEQ ID NO:373 and SEQ ID NO:374; SEQ ID NO:377 and SEQ IDNO:378; SEQ ID NO:381 and SEQ ID NO:382; SEQ ID NO:385 and SEQ IDNO:386; SEQ ID NO:389 and SEQ ID NO:390; SEQ ID NO:393 and SEQ IDNO:394; SEQ ID NO:397 and SEQ ID NO:398; SEQ ID NO:401 and SEQ IDNO:402; SEQ ID NO:405 and SEQ ID NO:406; and SEQ ID NO:409 and SEQ IDNO:410;
 35. A composition according to claim 34 wherein said variableheavy sequence has SEQ ID NO:397 and said variable light sequence hasSEQ ID NO:398.
 36. A composition according to claim 34 wherein saidvariable heavy sequence and said variable light sequence are linkedtogether to form an scFv domain.
 37. A composition according to claim 36wherein said composition further comprises an Fc region.
 38. Acomposition according to claim 37 wherein said Fc region is a variableFc region as compared to a wild-type human Fc region, wherein saidvariable Fc region has a an amino acid substitution selected from thegroup consisting of 239D, 239E, 236R, 330L, 332D, 332E, 239D/332E,239D/332E/330L, 267D, 267E, 328F, 328R, 267E/328F, 243L, 298A,236R/328R, 434A, 434S, 428L, 308F, 259I, 428L/434S, 259I/308F,436I/428L, 436I or V/434S, 436V/428L, 252Y, 252Y/254T/256E and259I/308F/428L.
 39. A composition according to claim 34 wherein saidcomposition has a structure selected from the group consisting of FIG.7A-7M, FIGS. 8B and 8D, FIG. 9B, FIGS. 10A-10E and FIGS. 11A-E, whereinthe Fc domain of said structure comprises a set of correspondingvariants selected from FIG.
 12. 40. A composition according to claim 34,wherein said composition has a structure selected from the groupconsisting of FIG. 7A-7M, FIGS. 8B and 8D, FIG. 9B, FIGS. 10A-10E andFIGS. 11A-E, wherein the Fc domain of said structure comprises a set ofcorresponding variants selected from FIG.
 13. 41. A compositionaccording to claim 34, wherein said composition has a structure selectedfrom the group consisting of FIG. 7A-7M, FIGS. 8B and 8D, FIG. 9B, FIGS.10A-10E and FIGS. 11A-E, wherein the Fc domain of said structurecomprises a set of corresponding variants selected from FIG.
 14. 42. Anucleic acid encoding a composition according to claim
 34. 43. Anexpression vector comprising a nucleic acid according to claim
 42. 44. Ahost cell comprising a nucleic acid encoding a composition according toclaim
 34. 45. A method of making a composition by providing a host cellaccording to claim 44 and culturing it under conditions wherein saidcomposition is expressed.
 46. A method according to claim 45 furthercomprising running said composition on an ion exchange column.
 47. Amethod of treating a patient in need thereof by administering acomposition according to claim 34.